Methods of making antibodies that bind Nogo

ABSTRACT

The present invention relates to the gene, Nogo, its encoded protein products, as well as derivatives and analogs thereof. Production of Nogo proteins, derivatives, and antibodies is also provided. The invention further relates to therapeutic compositions and methods of diagnosis and therapy.

This application is a continuation of U.S. patent application Ser. No.09/830,972 filed Sep. 24, 2001, which is the national stage ofinternational patent application no. PCT/US99/26160 filed Nov. 5, 1999,which claims benefit under 35 U.S.C.

119(e) of United States provisional application No. 60/107,446 filedNov. 6, 1998, all of which are incorporated herein by reference in theirentireties.

1. INTRODUCTION

The present invention relates to the gene, Nogo, and in particular toNogo, its encoded protein products, as well as derivatives and analogsthereof. Production of Nogo proteins, derivatives, and antibodies isalso provided. The invention further relates to therapeutic compositionsand methods of diagnosis and therapy.

2. BACKGROUND OF THE INVENTION

In the central nervous system (CNS) of higher vertebrates, regenerationof axons after injury is almost absent and structural plasticity islimited. Growth inhibitors associated with CNS myelin are likely to playan important role. This is evidenced by a monoclonal antibody (mAb),IN-1, that neutralizes a potent neurite growth inhibitory myelinprotein, thereby promoting long-distance axonal regeneration andenhancing compensatory plasticity following spinal cord or brain lesionsin adult rats.

A number of in vitro and in vivo observations have revealed a new aspectof neurite growth regulation which is the presence of repulsive andinhibitory signals and factors (Keynes and Cook, 1995, Curr. Opin.Neurosci. 5:75-82). Most of these signals seemed to be proteins orglycoproteins. A first breakthrough towards identification of thefactors was the purification and cDNA cloning of a chick brain-derivedgrowth cone collapse inducing molecule, Collapsin-1, now calledSemaphorin 3A.

A second group of repulsive guidance cues recently purified and clonedis now designated as Ephrins. They are ligands for the Eph receptortyrosine kinase family. Ephrin-A5 and Ephrin-A2 are expressed asgradients in the optic tectum of the chick embryo, and their ectopicexpression or deletion causes guidance errors of ingrowing retinalaxons. Like the Semaphorins, the Ephrin family has 15 to 20 members,each with a complex and dynamic expression in and outside of the nervoussystem. The functions of most of these molecules remain to be analyzed.

A third group of guidance cues which can repulse growing axons and isexpressed in the developing nervous system are the Netrins. Netrin hasbeen purified as a floor plate derived chemoattractant for commissuralaxons in early spinal cords, like its C. elegans ortholog unc-6.Netrin-1 turned out to have repulsive effects for certain types ofneurons depending on the type of receptor present on the target neuronalgrowth cones (Tessier-Lavigne et al., 1996, Science 274:1123-33).

Previously, a potent neurite growth inhibitory activity associated withadult CNS oligodendrocytes and myelin was reported by Canoni and Schwab(1988, J. Cell Biol. 106:1281-1288). A major constituent is a highmolecular weight membrane protein (NI-250, with a smaller component,NI-35, in rat) which was recently purified, and which is related to thesubject of the present invention, and is bound by the neutralizing mAb,IN-1 (Canoni and Schwab, 1988, J. Cell Biol. 106:1281-1288; U.S. Pat.Nos. 5,684,133; 5,250,414; PCT Publication WO 93/00427).

Myelin-associated neurite growth inhibitors play a crucial role inpreventing regeneration of lesioned CNS axons. When oligodendrocytedevelopment and myelin formation is blocked in chicken or rats, theregeneration permissive period following CNS lesions is prolonged.Indeed, myelin formation coincides in time with the end of thedevelopmental period where the CNS shows high structural plasticity anda high potential for regeneration.

NI-250 and NI-35 are likely to be major components of themyelin-associated growth inhibition as evidenced by in vivo applicationof IN-1 to spinal cord lesioned adult rats which induced regeneration ofcorticospinal axons over long distances and allowed motor and behaviorfunctional recovery especially with regard to locomotion. Similarexperiments on the optic nerve and the cholinergic septo-hippocampalpathway also demonstrated the in vivo relevance of the IN-1 recognizedantigen, NI-35/250 (Schnell and Schwab, 1990, Nature 343:269-272;Bregman et al., 1995, Nature 378:498-501).

Unlesioned fiber systems also respond to the neutralization of neuritegrowth inhibitors by IN-1. Recent experiments have conclusively shownthat following a selective corticospinal tract lesion (pyramidotomy),intact fibers sprout across the midline in the spinal cord and brainstemand establish a bilateral innervation pattern, accompanied by an almostfull behavioral recovery of precision movements in the presence of IN-1(Z'Graggen et al., 1998, J. Neuroscience 18(12):4744-4757).

Isolation of the gene that encodes the neurite growth inhibitory proteinprovides multiple opportunities for developing products useful inneuronal regeneration and in treatment of various neurologicaldisorders, including CNS tumor.

Citation of a reference hereinabove shall not be construed as anadmission that such reference is available as prior art to the presentinvention.

3. SUMMARY OF THE INVENTION

The present invention relates to nucleotide sequences of Nogo genes(human, rat and bovine Nogo and Nogo homologs of other species), andamino acid sequences of their encoded proteins, as well as derivatives(e.g., fragments) and analogs thereof. Nucleic acids hybridizable to orcomplementary to the foregoing nucleotide sequences are also provided.In a specific embodiment, the Nogo protein is a rat, bovine or humanprotein.

The invention also relates to a method of identifying genes whichinteract with Nogo.

Nogo is a gene provided by the present invention, identified by themethod of the invention, that both encodes and interacts with neuralgrowth regulatory proteins.

The invention also relates to Nogo derivatives and analogs of theinvention which are functionally active, i.e., they are capable ofdisplaying one or more known functional activities associated with anaturally occurring Nogo protein. For example, a major inhibitory regionbetween amino acids 542 to 722 have been identified. Such functionalactivities include, but are not limited to, neurite growth inhibition ofneural cells, spreading and migration of fibroblasts, or any cellexhibiting neoplastic growth, the ability to interact with or competefor interaction with neural growth regulatory proteins, antigenicitywhich is the ability to bind (or compete with Nogo for binding) to ananti-Nogo antibody, immunogenicity which is the ability to generateantibody which binds to Nogo. These antibodies having the potential toinduce neurite outgrowth or prevent dorsal root ganglia growth conecollapse by inhibiting the function of Nogo, and functional fragments orderivatives of Nogo, with the ability to inhibit neurite outgrowth.

The invention further relates to fragments (and derivatives and analogsthereof) of Nogo which comprise one or more domains of a Nogo proteinsuch as the acidic and proline rich amino terminus (e.g., at amino acids31 to 58 of SEQ ID NO:2), the highly conserved carboxy terminus, and twohydrophobic stretches of 35 and 36 amino acids length in rat Nogo, alsoin the carboxy terminus (e.g., at amino acids 988 to 1023, and at 1090to 1125 of SEQ ID NO:2).

Antibodies to the various Nogo, and Nogo derivatives and analogs, areadditionally provided. In particular, by way of example, two antibodieshave been derived, the first antibody, termed AS 472, was derived usingas immunogen a synthetic peptide corresponding to amino acids 623 to 640of SEQ ID NO:2, and the second antibody, termed AS Bruna, was generatedagainst the carboxy-terminus, amino acids 762 to 1163 of SEQ ID NO:2, ofNogo.

Methods of production of the Nogo proteins, derivatives and analogs,e.g., by recombinant means, are also provided.

The present invention also relates to therapeutic and diagnostic methodsand compositions based on Nogo proteins and nucleic acids. Therapeuticcompounds of the invention include but are not limited to Nogo proteinsand analogs and derivatives (including fragments) thereof; antibodiesthereto; nucleic acids encoding the Nogo proteins, analogs, orderivatives; and Nogo ribozymes or Nogo antisense nucleic acids.

The present invention also relates to therapeutic and diagnostic methodsand compositions based on Nogo proteins and nucleic acids and anti-Nogoantibodies. The invention provides for treatment of CNS and neuralderived tumors by administering compounds that promote Nogo activity(e.g., Nogo proteins and functionally active analogs and derivativesincluding fragments thereof; nucleic acids encoding the Nogo proteins,analogs, or derivatives, agonists of Nogo).

The invention also provides for treatment of diseases, disorders ordamage which ultimately result in damage of the nervous system; suchdiseases, disorders or damage include, but are not limited to, centralnervous system (CNS) trauma, (e.g. spinal cord or brain injuries),infarction, infection, malignancy, exposure to toxic agents, nutritionaldeficiency, paraneoplastic syndromes, and degenerative nerve diseases(including but not limited to Alzheimer's disease, Parkinson's disease,Huntington's Chorea, multiple sclerosis, amyotrophic lateral sclerosis,and progressive supra-nuclear palsy); by administering compounds thatinterfere with Nogo activity (e.g., a dominant negative Nogo derivative;antibodies to Nogo; anti-sense nucleic acids of Nogo; Nogo ribozymes orchemical groups that bind an active site of Nogo).

Animal models, diagnostic methods and screening methods forpredisposition to disorders, and methods to identify and evaluate Nogoagonists and antagonists, are also provided by the invention.

The invention also provides a purified protein comprising a fragment ofa Nogo protein comprising an amino acid sequence selected from the groupconsisting of residues 31-57 depicted in FIG. 2 a (SEQ ID NO:2),residues 11-191 depicted in FIG. 14 (SEQ ID NO:32), residues 988-1023depicted in FIG. 2 a (SEQ ID NO:2), residues 1090-1125 depicted in FIG.2 a (SEQ ID NO:2), residues 994-1174 depicted in FIG. 13 (SEQ ID NO:29),residues 977-1012 depicted in FIG. 13 (SEQ ID NO:29), and residues1079-1114 depicted in FIG. 13 (SEQ ID NO:29).

3.1 DEFINITIONS

As used herein, underscoring or italicizing the name of a gene shallindicate the gene, in contrast to its encoded protein product which isindicated by the name of the gene in the absence of any underscoring oritalicizing. For example, “Nogo” shall mean the Nogo gene, whereas“Nogo” shall indicate the protein product of the Nogo gene.

4. DESCRIPTION OF THE FIGURES

FIG. 1 a-1 b: (a) Nogo cDNA clones: CWP1-3 is a bovine cDNA cloneisolated from the screening of a bovine spinal cord white matter cDNAlibrary with degenerated oligonucleotides MSC5-8 (pooled) and MSC9.Complementary RNA derived from this clone was used for subsequent ratcDNA library screening. Oli3 and Oli18 are isolated from an oligod(T)-primed rat oligodendrocyte library. R1-3U21, RO18U1 and RO18U37-3are isolated from a hexanucleotides-primed rat brain stern/spinal cordlibrary (Stratagene). The positions of the 6 bovine NI220 (bNI220)peptide sequences are indicated on CWP1-3 and R13U21. Sequences at thejunctions of different exons are marked on top of each clone. Thequestion marks indicated on RO18U1 identify the sequence on this clonewhich does not match sequences from any other Nogo clones. RO18U37-3 wassequenced-only from the 5′-end, and the unsequenced portion isrepresented by dots. (b) Schematics demonstrating the hypotheticalmechanism for the generation of three Nogo transcripts. P1 and P2represent the putative location of the alternative promoters. Theminimum number of three exons is required for generating the threetranscripts as shown schematically, although each exon could potentiallybe split into multiple exons.

FIG. 2A1 to 2A4: Nucleotide (SEQ ID NO:1) and amino acid sequences (SEQID NO:2) of Nogo transcript A (sequence generated by connectingRO18U37-3, Oli18, and R1-3U21 cDNA sequences). Oval box: presumedinitiation codon; underlined with dots: acidic stretch; □: potential PKCsites; Δ: potential casein kinase II sites; thick underline: carboxyterminal hydrophobic regions and potential transmembrane domains; thinunderline: potential N-glycosylation sites. 2B: Peptide sequencecomparison between sequenced, purified bovine NI220 (bNI220; SEQ IDNOS:3-8), and the corresponding bovine (SEQ ID NOS:9-14) and rat (SEQ IDNOS:15-20) sequences translated from rat and bovine cDNAs. Rat andbovine amino acid sequences, which do not match the bNI220 peptidesequences, are in lower case.

FIG. 3 a-3 b: (a) Amino acid sequence comparison of the carboxy terminal180 amino acid common regions of NSP (human; SEQ ID NO:21), S-REX(rat)(SEQ ID NO:22), CHS-REX (chicken; SEQ ID NO:23), NOGOBOV (bovine;SEQ ID NO:24), NOGORAT (rat; SEQ ID NO:25), a C. elegans EST clone(WO6A7A; SEQ ID NO:26), and a D. melanogaster EST clone (US51048; SEQ IDNO:27). (b) Evolutionary conservation of the two hydrophobic regions.Percent similarities within and across species of the common hydrophobicregions are shown. Shaded letters: conserved amino acids.

FIG. 4 a-4 c: (a) Northern hybridization of various tissues with theNogo common probe. The common probe contains transcript A sequencebetween nucleotides 2583-4678. ON, optic nerve; SC, spinal cord; C,cerebral cortex; DRG, dorsal root ganglia; SN, sciatic nerve; PC12, PC12cell line. (b) Northern hybridization of spinal cord and PC12 cells RNAswith an exon 1-specific probe (left panel) and of hindbrain (HB) andskeletal muscle (M) RNAs with an exon 2 specific probe (right panel).(c) Northern hybridization with the Nogo common probe. K, kidney; B,cartilage (from breastbone); Sk, skin; M, skeletal muscle; Lu, lung; Li,liver; Sp, spleen. The three major transcripts are marked (4.6 kilobases(kb), 2.6 kb, and 1.7 kb). Δ: a diffuse but consistent band about 1.3 kbin size.

FIG. 5 a-5 f: In situ hybridization of adult rat spinal cord andcerebellum sections. (a, d) Rows of oligodendrocytes (OL) in spinal cordand cerebellum white matter, respectively, can be seen labeled by theNogo antisense “common” riboprobe. This is very similar to the signalsdetected when a consecutive spinal cord section was hybridized to anantisense plop riboprobe (b). (c) Neurons in grey matter (GM) were alsolabeled by the Nogo antisense “common” riboprobe. WM: white matter.Bright field and fluorescent view, respectively, of a cerebellum sectiondouble labeled with the Nogo antisense “common” riboprobe (e) and of ananti-GFAP antibody (f). Purkinje cells (double arrowheads) are stronglylabeled with the Nogo probe, while astrocytes (arrowheads, black andwhite) are negative. A few cells in the granular cell layer (Gr) arealso labeled with the Nogo probe, m: molecular layer. Scale bar: a, b,d-f: 50 p.m; c: 280 p.m.

FIG. 6 a-6 i: In situ hybridization of optic nerves at differentpostnatal days (a,f: P0; b,g: P3; c,h: P7; d,e,i: P22) with either Nogoor plp (antisense or sense) probes. (a-d) Nogo antisense probe; (e) Nogosense probe; (g-i) plp antisense probe; (f) plp sense probe. Nogoexpression in oligodendrocyte precusors can be detected as early as P0,while plp expression was only beginning to be detectable in P3 opticnerves close to the chiasm (g).

FIG. 7: AS Bruna and AS 472 both recognize a myelin protein of about 200kD. Rat myelin extract and bovine q-pool were prepared according toSpillmann et al, 1998, J. Biol. Chem., 273(30):19283-19293. AS Bruna andAS 472 each recognized a 200 kD band as well as several lower bands inbovine myelin, which may be breakdown products of bNI220. AS Brunastained a band 200 kD in rat myelin. I: AS Bruna; P: AS Bruna, preimmuneserum; E: AS 472 affinity purified.

FIGS. 8 a-8 i: Immunohistochemistry on rat spinal cord and cerebellumusing IN-1 (a-e), AS Bruna (d-f), and AS 472 (g-i), as indicated at theleft of each panel. A strong myelin staining was observed in bothtissues with all three antibodies when the frozen sections were fixedwith ethanol/acetic acid (a, b, d, e, g, h). Treatment of the sectionswith methanol abolished the myelin staining except for oligodendrocytecell bodies (arrows; c, f, i). Arrowheads: Purkinje cells, WM, whitematter; GM, grey matter, DR: dorsal root; Gr, granular cell layer; m,molecular layer. Scale bar: a, d, g: 415 μm; b, c, e, f, h, i: 143 μm.

FIG. 9 a-9 d: Neutralizing activity of AS 472 and AS Bruna in differentbioassays. (a) the NIH 3T3 fibroblasts were plated on cell culturedishes coated with q-pool and pre-treated with IN-1, AS Bruna, AS 472 orthe corresponding pre-immune sera. Both polyclonal sera showed even aslightly better neutralization of the inhibitory substrate than IN-1.The pre-immune sera had no significant effect on the spreading of theNIH 3T3 cells. Addition of an excess of the peptide (P472) that was usedto raise AS 472 competed the neutralizing activity whereas an unspecificpeptide (Px) had no effect. (b) Pre-treatment of the inhibitorysubstrate with AS Bruna or AS 472 resulted in DRG neurite outgrowthcomparable to what can be observed on a laminin substrate. Examples forneurite outgrowth from DRG on q-pool pre-treated with PBS (c; score=0)and pretreated with AS Bruna (d; score=4).

FIG. 10 a-10 d: Injection of optic nerve explants with AS 472 results iningrowth of axons. (a) Pairs of adult rat optic nerves were dissected,injected with AS 472 or preimmune serum and placed into chamber culturessuch that one end of the nerves was in contact with dissociated P0 ratDRG neurons. (b) After two weeks in vitro, EM sections of the nerveswere taken at 3.5 mm from the cut site (arrows in A) and systematicallyscreened for intact axons (3 experiments). (c) Regenerated axon bundles(arrows) grow through degenerating AS 472 injected optic nerve. (d)Regenerating axons in contact with myelin. Magnification: c, 12,000×; d,35,000×.

FIG. 11 a-11 c: Recombinant Nogo A expression in transfected COS cells.(a) Western blot showing immunoreactivity of AS Bruna to recombinantNogo A (lane 2) and endogenous Nogo A from primary cultured ratoligodendrocytes (lane 3). The mobilities of these two proteins arevirtually identical at about 200 kD on a 5% denaturing SDS gel. Acontrol LacZ construct transfected sample (lane 1) showed noimmunoreactivity with AS Bruna. The same blot was also probed withanti-myc antibody, 9E10, as indicated. The band that reacted with ASBruna also reacted with the anti-myc tag antibody, 9E10 (lane 5), whilethe endogenous Nogo A did not (lane 6). The LacZ control transfectionsample showed the expected band at about 118 kD (lane 4). COS cellstransiently transfected with a Nogo A construct were double stained withAS Bruna (b) and IN-1 (c). Cells positively stained with AS Bruna werealso positive with IN-1.

FIG. 12A to 12D: The nucleotide sequence (SEQ ID NO:28) of the bovineNogo cDNA.

FIG. 13A-13B: The amino acid sequence of rat Nogo A (SEQ ID NO:2)aligned with the theoretical amino acid sequence of human Nogo (SEQ IDNO:29). The human Nogo amino acid sequence was derived from aligningexpressed sequence tags (EST) to the rat Nogo sequence and translatingthe aligned human ESTs using the rat Nogo as a guiding template.

FIG. 14A-14C: Rat Nogo C nucleic acid (SEQ ID NO:31) sequence and itscorresponding amino acid sequence (SEQ ID NO:32).

FIG. 15 a-15 e: Nogo A is present on the oligodendrocyte plasmamembrane, as demonstrated by immunocytochemistry and cell surfacebiotinylation of oligodendrocytes in culture.

Immunocytochemistry (a-d): Oligodendrocytes from optic nerves of P10rats were dissociated and cultured for 2 days. Staining of live cellswith mAb IN-1 (a) or AS 472 (c) showed immunoreactivity onoligodendrocyte cell bodies and processes. In the presence of thecompeting peptide P472, AS 472 showed only background labeling (all celltypes) (d). Similar non-specific staining was seen when primaryantibodies were omitted (b). Evaluation: Number-coded dishes wererandomly mixed and classified by 3 independent observers. 8/10 disheswere correctly classified AS 472-positive, mAb IN-1-positive or controlsby all three observers.

Biotinylation (e): Rat P4 whole brain cultures enriched inoligodendrocytes were cell surface biotinylated with a membraneimpermeable reagent after seven days in culture. Subsequently, cellhomogenates were treated with streptavidin-Dynabeads. Precipitate (Ppt)and supernatant (sup) were blotted with AS472; they showed a distinctprotein pattern: Cell surface Nogo A found in the precipitate showed ahigher apparent molecular weight than intracellular Nogo A. This shiftis probably due to glycosylation. The luminal ER protein BiP and thelarge majority of β-tubulin could only be found in the intracellularfraction.

FIG. 16 a-16 j: Functional assays show the presence of Nogo A on thecell membrane of oligodendrocytes. Pre-incubation of optic nervecultures with AS 472 (a, b) allowed the NIH 3T3 fibroblasts to spreadover the highly branched oligodendrocytes which are outlined byimmunofluorescent staining for GalC (01 antibody) (a). Arrows in thecorresponding phase contrast image (b) indicate the NIH 3T3 fibroblastsspreading on top of the oligodendrocytes. (c, d): When AS 472 was addedtogether with P472, the NIH 3T3 fibroblasts strictly avoided theterritories of the GalC-positive oligodendrocytes (arrowheads) (Caroniand Schwab, 1988 Neuron 1:85-96). (e, f): In the presence of AS 472, P0rat dissociated DRG neurons were able to extend neurites over theterritory of highly branched oligodendrocytes (arrows in f). (g, h): Thepeptide P472 efficiently competed the neutralizing activity of AS 472:the neurites completely avoided the oligodendrocytes. AS 472 used inthese experiments was generated against the rat 472 peptide sequence.(i, j): Quantification of these results (as described in methods)demonstrated the strong neutralizing activity of AS 472 in both types ofassays. Scale bar: 40 μm.

FIG. 17 a-17 e: Recombinant Nogo A is an inhibitory substrate and itsinhibitory activity is neutralized by mAb IN-1. RecNogo A enrichedextracts from a stable CHO-Nogo A cell line, or β-galactosidase,isolated in parallel from the stable CHO-LacZ cell line, were coated forthe NIH 3T3 fibroblast spreading and DRG neurite outgrowth assays. (a)Silver gel of myc-his-tagged recLacZ (lane 1) and recNogo A (lane 2)shows the Nogo A band at 180 kD. The identity of the Nogo A band wasconfirmed by Western blot incubated with AS Bruna (lane 3) and ananti-myc antibody 9E10 (lane 4). (b) RecNogo A coated dishes wereclearly inhibitory to the NIH 3T3 spreading. Pre-incubation with mAbIN-1 or AS Bruna resulted in a highly significant (p<0.01)neutralization of inhibitory activity. The control 1 gM mAb O1 andpre-immune serum were ineffective. CHO-LacZ extract had a partialinhibitory effect on the NIH 3T3 cells, probably due to endogenous CHOproteins. This inhibitory activity was not influenced by pre-incubationwith antibodies.

(c) For DRG neurite outgrowth assays, the same protein material as in(b) was mixed with laminin and coated. RecNogo A had a very potentinhibitory effect on neurite outgrowth of dissociated DRG in adose-dependent manner. The activity was neutralized by mAb IN-1(p<0.001), but not by control mAb 01. Protein material isolated fromCHO-LacZ cells was not inhibitory at any of the concentrations used, nordid incubation with antibodies have any effect on neurite outgrowth.Examples for scoring are shown in (d): 1 μg recNogo A, no or shortneurites (arrows) score: 2, and in (e): 1 μg CHO-LacZ, long, branchedneurites (arrowheads) score: 5-6. Statistical analysis was performedwith two-tailed Student's t test. Scale bar: 280 μm.

FIG. 18: Functional Analysis of Nogo Deletion Mutants. The followingdeletion constructs encoding fusion proteins containing fragments ofNogo or truncated portions of Nogo (as listed below) were generated asdescribed in Section 6.2.7 hereinbelow.

-   Nogo-A: His-tag/T7-tag/vector/Nogo-A seq. aa1-1162.-   Nogo-B: His-tag/T7-tag/vector/Nogo-A seq. aa1-171+975-1162-   Nogo-C: His-tag/T7-tag/Nogo-C N-terminus (11 aa)+Nogo-A seq. aa    975-1162-   NiAext: His-tag/T7-tag/vector/Nogo-A seq. aa1-974/T7-tag-   NiR: His-tag/T7-tag/vector/Nogo-A seq. aa1-171/vector-   NiG: His-tag/T7-tag/Nogo-A seq. aa 172-974/His-tag-   EST: His-tag/T7-tag/Nogo-A seq. aa 760-1162-   NiG-D1: His-tag/T7-tag/Nogo-A seq. aa172-908/vector-   NiG-D2: His-tag/T7-tag/Nogo-A seq. aa 172-866/His-tag-   NiG-D3: His-tag/T7-tag/Nogo-A seq. aa 172-723/His-tag-   NiG-D4: His-tag/T7-tag/Nogo-A seq. aa 172-646/vector-   NiG-D5: His-tag/T7-tag/Nogo-A seq. aa 291-646/His-tag-   NiG-D7: His-tag/T7-tag/Nogo-A seq. aa 172-234+292-974/His-tag-   NiG-D8: His-tag/T7-tag/Nogo-A seq. aa 172-628-   NiG-D9: His-tag/T7-tag/Nogo-A seq. aa 172-259+646-974/His-tag-   NiG-D10: His-tag/T7-tag/Nogo-A seq. aa 291-974/His-tag-   NiG-D14: His-tag/T7-tag/Nogo-A seq. aa 172-259-   NiG-D15: His-tag/T7-tag/Nogo-A seq. aa 172-189+491-974/His-tag-   NiG-D16: His-tag/T7-tag/Nogo-A seq. aa 172-189+619-974/His-tag-   NiG-D17: His-tag/T7-tag/Nogo-A seq. aa 172-189+257-974/His-tag-   NiG-D18: His-tag/T7-tag/Nogo-A seq. aa 172-189+261-974/His-tag-   NiG-D20: His-tag/T7-tag/Nogo-A seq. aa 542-722/His-tag    The amino acid (aa) numbers are based on rat Nogo A amino acid    sequence numbering (SEQ ID NO: 2) starting with the first    methionine. The His-tag and T7-tag consist of 34 amino acids. The N-    and C-terminal vector sequences are derived from the expression    vector pET28.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to nucleotide sequences of Nogo genes, andamino acid sequences of their encoded proteins. The invention furtherrelates to fragments and other derivatives, and analogs, of Nogoproteins. Nucleic acids encoding such fragments or derivatives are alsowithin the scope of the invention. The invention provides Nogo genes andtheir encoded proteins of many different species. The Nogo genes of theinvention include human, rat and bovine Nogo and related genes(homologs) in other species. The bovine subsequences disclosed inSpillman et al., 1998, J. Biol. Chem. 273:19283-19293, are not claimedas part of the present invention. In specific embodiments, the Nogogenes and proteins are from vertebrates, or more particularly, mammals.In a preferred embodiment of the invention, the Nogo genes and proteinsare of human origin. Production of the foregoing proteins andderivatives, e.g., by recombinant methods, is provided.

The Nogo gene as provided by the present invention, encompasses nucleicacid molecules encoding three isoforms of Nogo; namely Nogo A, Nogo Band Nogo C. Reference to the gene “Nogo” shall include nucleic acidmolecules encoding all three isoforms unless otherwise specified.Likewise, reference to Nogo protein shall include all three isoforms ofNogo unless otherwise specified. Nogo proteins of the invention canprevent regeneration of neurons in the spinal cord or brain (i.e.non-permissive substrate properties), inhibit dorsal root ganglianeurite outgrowth, induce dorsal root ganglia growth cone collapse,block NIH 3T3 cell spreading, block PC12 neurite outgrowth, etc.

The Nogo proteins, fragments and derivatives thereof are free of allcentral nervous system myelin material; in particular, they are free ofall central nervous system myelin material with which the Nogo proteinis naturally associated. Such material may include other CNS myelinproteins, lipids, and carbohydrates. The Nogo proteins, fragments andderivatives thereof of the invention are also preferably free of thereagents used in purification from biological specimens, such asdetergents.

In a specific embodiment, the invention provides recombinant Nogoproteins, fragments and derivatives thereof as prepared by methods knownin the art, such as expressing the Nogo gene in a genetically engineeredcell.

The invention also relates to Nogo derivatives and analogs of theinvention which are functionally active, i.e., they are capable ofdisplaying one or more known functional activities associated with afull-length (wild-type) Nogo protein. Such functional activities includebut are not limited to the ability to interact (or compete for binding)with neural growth regulatory proteins, antigenicity [ability to bind(or compete with Nogo for binding) to an anti-Nogo antibody],immunogenicity (ability to generate antibody which binds to Nogo),preventing regeneration of neurons in the spinal cord or brain,conferring to a substrate the property of restricing growth, spreading,and migration of neural cells, and neoplastic cells, inhibiting dorsalroot ganglia neurite outgrowth, inducing dorsal root ganglia growth conecollapse, blocking NIH 3T3 cell spreading in vitro, blocking PC12neurite outgrowth, restricting neural plasticity, etc.

The invention further relates to fragments (and derivatives and analogsthereof) of Nogo which comprise one or more domains of the Nogo protein.

Antibodies to Nogo, its derivatives and analogs, are additionallyprovided.

The present invention also relates to therapeutic and diagnostic methodsand compositions based on Nogo proteins and nucleic acids and anti-Nogoantibodies. The invention provides for treatment of disorders of growthregulated cells or organs by administering compounds that promote Nogoactivity (e.g., Nogo proteins and functionally active analogs andderivatives (including fragments) thereof; nucleic acids encoding theNogo proteins, analogs, or derivatives, agonists of Nogo).

The invention also provides methods of treatment of damage or disorderof the nervous system by administering compounds that antagonize, orinhibit, Nogo function (e.g., antibodies, Nogo antisense nucleic acids,Nogo antagonist derivatives).

Animal models, diagnostic methods and screening methods forpredisposition to disorders are also provided by the invention.

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections whichfollow.

5.1 Isolation of Nogo Genes

The invention relates to the nucleotide sequences of Nogo genes ornucleic acids. In one embodiment, Nogo nucleic acids comprise the ratcDNA sequence of FIG. 2 a (SEQ ID NO:1) identified as Nogo A as depictedin FIG. 1 b, or the coding regions thereof, or nucleotide sequencesencoding a Nogo protein of 1163 amino acids in length or any functionalfragment or derivative thereof (e.g., a protein having the sequence ofSEQ ID NO:2, as shown in FIG. 2 a).

In another embodiment, Nogo nucleic acids comprise the nucleotidesequence encoding Nogo B, whereas the Nogo B protein is equivalent tothe amino terminal 172 amino acids fused to the carboxy terminal 188amino acids of Nogo A, resulting in a truncated 360 amino acid protein.The transcripts for Nogo B arise as a result of alternative splicingwhich removes the intervening nucleotide coding sequence.

In yet another embodiment of the present invention, Nogo nucleic acidscomprise the nucleotide sequences encoding Nogo C, whereas the Nogo Cprotein contains 11 amino acids at its amino terminus which are notpresent in Nogo A, and the carboxy terminal 188 amino acids of Nogo Aand B. The Nogo C protein has 199 amino acids. The transcript encodingNogo C is the result of transcription from an alternative Nogo promoter.

In yet another specific embodiment, the present invention providesbovine Nogo nucleic acid sequences (SEQ ID NO:28).

In yet another specific embodiment, the instant invention provides thenucleotide sequences encoding human Nogo, and fragments of human Nogoproteins, including the human equivalents to rat Nogo A, Nogo B and NogoC. The human Nogo nucleic acid sequence is elucidated using the rat NogoA transcript as a template and splicing together human expressedsequence tags (EST) to reveal a continuous nucleotide sequence. The ratand bovine amino acid sequences of Nogo also provided information on theproper translational reading frame such that an amino acid sequence ofhuman Nogo is deduced. The instant invention also provides amino acidsequences of fragments of the human Nogo gene.

The invention also provides purified nucleic acids consisting of atleast 8 nucleotides (i.e., a hybridizable portion) of a Nogo sequence;in other embodiments, the nucleic acids consist of at least 25(continuous) nucleotides, 50 nucleotides, 100 nucleotides, 150nucleotides, 20 nucleotides, 500 nucleotides, 700 nucleotides, or 800nucleotides of a Nogo sequence, or a full-length Nogo coding sequence.In another embodiment, the nucleic acids are smaller than 35, 200 or 500nucleotides in length. Nucleic acids can be single or double stranded.The invention also relates to nucleic acids hybridizable to orcomplementary to the foregoing sequences. In specific aspects, nucleicacids are provided which comprise a sequence complementary to at least10, 25, 50, 100, or 200 nucleotides or the entire coding region of aNogo gene.

In a specific embodiment, a nucleic acid which is hybridizable to a Nogonucleic acid (e.g., having sequence SEQ ID NO:2; FIG. 2 a), or to anucleic acid encoding a Nogo derivative, under conditions of lowstringency is provided. By way of example and not limitation, proceduresusing such conditions of low stringency are as follows (see also Shiloand Weinberg, 1981, Proc. Natl. Acad. Sci. USA 78:6789-6792): Filterscontaining DNA are pretreated for 6 h at 40° C. in a solution containing35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1%Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizationsare carried out in the same solution with the following modifications:0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10%(wt/vol) dextran sulfate, and 5-20×10⁶ cpm ³²P-labeled probe is used.Filters are incubated in hybridization mixture for 18-20 h at 40° C.,and then washed for 1.5 h at 55° C. in a solution containing 2×SSC, 25mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution isreplaced with fresh solution and incubated an additional 1.5 h at 60° C.Filters are blotted dry and exposed for autoradiography. If necessary,filters are washed for a third time at 65-68° C. and reexposed to film.Other conditions of low stringency which may be used are well known inthe art (e.g., as employed for cross-species hybridizations asdemonstrated in the example in Section 6.1.1).

In another specific embodiment, a nucleic acid which is hybridizable toa Nogo nucleic acid under conditions of high stringency is provided. Byway of example and not limitation, procedures using such conditions ofhigh stringency are as follows: Prehybridization of filters containingDNA is carried out for 8 h to overnight at 65° C. in buffer composed of6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll,0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters arehybridized for 48 h at 65° C. in prehybridization mixture containing 100μg/ml denatured salmon sperm DNA and 5-20×10⁶ cpm of ³²P-labeled probe.Washing of filters is done at 37° C. for 1 h in a solution containing2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by awash in 0.1×SSC at 50° C. for 45 min before autoradiography. Otherconditions of high stringency which may be used are well known in theart.

In another specific embodiment, a nucleic acid, which is hybridizable toa Nogo nucleic acid under conditions of moderate stringency is provided.For example, but not limited to, procedures using such conditions ofmoderate stringency are as follows: Filters containing DNA arepretreated for 6 h at 55° C. in a solution containing 6×SSC, 5×Denhart's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA.Hybridizations are carried out in the same solution and 5-20×10⁶ cpm³²P-labeled probe is used. Filters are incubated in hybridizationmixture for 18-20 h at 55° C., and then washed twice for 30 minutes at60° C. in a solution containing 1×SSC and 0.1% SDS. Filters are blotteddry and exposed for autoradiography. Other conditions of moderatestringency which may be used are well-known in the art. Washing offilters is done at 37° C. for 1 h in a solution containing 2×SSC, 0.1%SDS. Such stringency conditions are suitable for isolating nucleic acidmolecules comprising Nogo gene sequences in another species, e.g., usingthe rat or bovine Nogo cDNA clones as probe to isolate the human NogocDNA.

A number of human expressed sequence tags (ESTs) reported in publishednucleic acid sequence databases display a high degree of sequenceidentity when compared to segments of the Nogo gene sequences of theinvention. The following preliminary list of human ESTs were identifiedand are listed by their Genbank accession numbers: AA158636 (SEQ IDNO:35), AA333267 (SEQ ID NO:36), AA081783 (SEQ ID NO:37), AA167765 (SEQID NO:38), AA322918 (SEQ ID NO:39), AA092565 (SEQ ID NO:40), AA081525(SEQ ID NO:41), and AA081840 (SEQ ID NO:42) using ENTREZ NucleotideQuery. Prior to the present invention, none of the above-identified ESTshad been characterized with respect to the amino acid sequences theseESTs may encode in vivo. Nothing was known about the function of theproteins comprising the predicted amino acid sequences of the humanESTs. Furthermore, an EST, such as AA158636, aligning with the 5′ end ofrat Nogo cDNA and another EST, such as AA081840, aligning with the 3′end of rat cDNA, are not overlapping and would not be perceived to bepart of the same human cDNA sequence.

Based on the Nogo gene sequences of the present invention, it isbelieved that these human ESTs represent portions of the human Nogo genethat are expressed in the tissue from which the ESTs were obtained.Accordingly, the present invention encompasses nucleic acid moleculescomprising two or more of the above-identified human ESTs. The ESTs maybe expressed in the same human tissue, or in different human tissues.Preferably, the nucleic acid molecules of the invention comprise thenucleotide sequences of at least two human ESTs which are notoverlapping with respect to each other, or which do not overlap a thirdor more human EST.

Since the above-identified human ESTs are now identified as fragments ofthe human Nogo gene due to the cloning of bovine and rat Nogo nucleicacids, it is contemplated that the human ESTs have similar functionsrelative to the other Nogo nucleic acid molecules in various methods ofthe invention, such as but not limited to, for example, the expressionof human Nogo polypeptides, hybridization assays, and inhibition of Nogoexpression as antisense nucleic acid molecules, etc.

Moreover, the present invention provides and includes the predictedamino acid sequence of the human Nogo protein, and fragments thereof. Asshown in FIG. 13, the amino acid sequence of rat Nogo protein (FIG. 2 a;SEQ ID NO:2) is aligned with the predicted amino acid sequence of humanNogo protein (FIG. 13; SEQ ID NO:29). Accordingly, the present inventionencompasses human Nogo proteins comprising the predicted amino acidsequence of human Nogo, FIG. 13 and SEQ ID NO:29, or a subsequence ofthe predicted amino acid sequence of human Nogo, consisting of at least6 amino acid residues, or one or more of the following predicted aminoacid sequences of human Nogo fragments: MEDLDQSPLVSSS (Human Nogo,corresponding to amino acids 1-13 with SEQ ID NO:43), KIMDLKEQPGNTISAG(Human Nogo, corresponding to amino acids 187-203 with SEQ ID NO:44),KEDEVVSSEKAKDSFNEKR (Human Nogo, corresponding to amino acids 340-358with SEQ ID NO:45), QESLYPAAQLCPSFEESEATPSPVLPDIVMEAPLNSAVPSAGASVIQPSS(Human Nogo, corresponding to amino acids 570-619 with SEQ ID NO:46).Naturally occurring human Nogo and recombinant human Nogo, and fragmentsthereof having an amino acid sequence substantially similar to theabove-described amino acid sequences and able to be bound by an antibodydirected against a Nogo protein are within the scope of the invention.

The present invention further provides nucleic acid molecules thatencodes a human Nogo protein having an amino acid sequence substantiallysimilar to the amino acid sequence as shown in FIG. 13 (FIG. 13; SEQ IDNO:29). In specific embodiments, nucleic acid molecules encodingfragments of human Nogo protein having an amino acid sequencesubstantially similar to the amino acid sequence as shown in FIG. 13(SEQ ID NO:29) are also contemplated with the proviso that such nucleicacid molecules do not comprise the nucleotide sequence of theabove-identified human ESTs.

An amino acid sequence is deemed to be substantially similar to thepredicted amino acid sequence of human Nogo protein when more than 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% of the amino acidresidues in the two molecules are identical when a computer algorithm isused in which the alignment is done by a computer homology program knownin the art, for example a BLAST computer searching (Altschul et al.,1994, Nature Genet. 6:119-129) is used.

By way of example and not limitation, useful computer homology programsinclude the following: Basic Local Alignment Search Tool (BLAST) (homepage of the National Center for Biotechnology Information) (Altschul etal., 1990, J. of Molec. Biol., 215:403-410, “The BLAST Algorithm;Altschul et al., 1997, Nuc. Acids Res. 25:3389-3402) a heuristic searchalgorithm tailored to searching for sequence similarity which ascribessignificance using the statistical methods of Karlin and Altschul 1990,Proc. Nat'l Acad. Sci. USA, 87:2264-68; 1993, Proc. Nat'l Acad. Sci. USA90:5873-77. Five specific BLAST programs perform the following tasks:

1) The BLASTP program compares an amino acid query sequence against aprotein sequence database.

2) The BLASTN program compares a nucleotide query sequence against anucleotide sequence database.

3) The BLASTX program compares the six-frame conceptual translationproducts of a nucleotide query sequence (both strands) against a proteinsequence database.

4) The TBLASTN program compares a protein query sequence against anucleotide sequence database translated in all six reading frames (bothstrands).

5) The TBLASTX program compares the six-frame translations of anucleotide query sequence against the six-frame translations of anucleotide sequence database.

As will be understood by those skilled in the art, the TBLASTN programis particularly useful to identify nucleic acids with a desired percentidentity and the BLASTP program is particularly useful to identify aminoacid sequences with a desired percent identity.

Smith-Waterman (database: European Bioinformatics Institute(Smith-Waterman, 1981, J. of Molec. Biol., 147:195-197) is amathematically rigorous algorithm for sequence alignments.

FASTA (see Pearson et al., 1988, Proc. Nat'l Acad. Sci. USA,85:2444-2448) is a heuristic approximation to the Smith-Watermanalgorithm. For a general discussion of the procedure and benefits of theBLAST, Smith-Waterman and FASTA algorithms, see Nicholas et al., 1998,“A Tutorial on Searching Sequence Databases and Sequence ScoringMethods” (home page of the Pittsburgh Supercomputing Center) andreferences cited therein.

The uses of the predicted amino acid sequences of human Nogo, or thenucleotide sequences of human ESTs, including degenerate sequencesencoding the predicted amino acid sequence of human Nogo, for isolatingor identifying the human Nogo gene, fragments, naturally occurringmutants and variants thereof, is within the scope of the invention. Suchuses which will be known to one of skill in the art include but are notlimited to using the information to prepare nucleic acid probes for DNAlibrary screening, DNA amplification, genetic screening of the humanpopulation, and to prepare synthetic peptides for making antibodies.Detailed description of some of such uses are provided herein in latersections.

Nucleic acids encoding derivatives and analogs of Nogo proteins, andNogo antisense nucleic acids are additionally provided. As is readilyapparent, as used herein, a “nucleic acid encoding a fragment or portionof a Nogo protein” shall be construed as referring to a nucleic acidencoding only the recited fragment or portion of the Nogo protein andnot the other contiguous portions of the Nogo protein as a continuoussequence. In this context, a portion means one or more amino acids.

Fragments of Nogo nucleic acids comprising regions conserved between(with homology to) other Nogo nucleic acids, of the same or differentspecies, are also provided. Nucleic acids encoding one or more Nogodomains are provided in FIG. 2 a, for example, the conserved carboxyterminal domain of rat Nogo, which has about 180 amino acids, and isencoded by the last 540 nucleotides of the coding sequence prior to thestop codon. The nucleotide and amino acid sequences of two hydrophobicdomains within the conserved carboxy terminus domain, i.e., from aminoacids 988 to 1023, and from amino acids 1090 to 1125, in rat Nogo A, arealso provided. The nucleotide and amino acid sequences of the aminoterminal acidic domain of rat Nogo A, from residues 31 to 58, are alsoprovided.

To perform functional analysis of various regions of Nogo, a series ofdeletions in the Nogo gene has been generated and cloned into anexpression vector by recombinant DNA techniques and expressed as afusion protein. Nucleic acids that encode a fragment of a Nogo proteinare provided, e.g., nucleic acids that encode amino acid residues 1-171,172-974, 259-542, 542-722, 172-259, 722-974, or 975-1162 of SEQ ID NO:2, or combinations thereof; and nucleic acids that encode amino acidresidues 1-131, 132-939, 206-501, 501-680, 132-206, 680-939, and940-1127 of SEQ ID NO:29, or combinations thereof. Some of the deletionconstructs comprises truncated portions of Nogo and additionalnucleotide sequences encoding a hexahistidine tag and/or a T7-tag.Nucleic acids encoding truncated Nogo proteins that lacks amino acidresidues 172-259, amino acid residues 974-1162, or amino acid residues172-259 and 974-1162, of SEQ ID NO:2 but otherwise comprises theremainder of SEQ ID NO: 2; or amino acid residues 132-206, amino acidresidues 939-1127, or amino acid residues 132-206 and 939-1127, of SEQID NO:29 but otherwise comprises the remainder of SEQ ID NO:29, areprovided. The structure of exemplary deletion constructs are shown inFIG. 18. The deletion constructs produce fragments or truncatedportion(s) of Nogo when introduced into a cell. The biologicalactivities of these mutants were tested in various functional assays asdescribed in Table 2 in Section 6.2.7.

For expression cloning (a technique commonly known in the art), anexpression library is constructed by methods known in the art. Forexample, mRNA (e.g., human) is isolated, cDNA is made and ligated intoan expression vector (e.g., a bacteriophage derivative) such that it iscapable of being expressed by the host cell into which it is thenintroduced. Various screening assays can then be used to select for theexpressed Nogo product. In one embodiment, anti-Nogo antibodies can beused for selection.

In another embodiment, polymerase chain reaction (PCR) is used toamplify the desired sequence in a genomic or cDNA library, prior toselection. Oligonucleotide primers representing known Nogo sequences canbe used as primers in PCR. In a preferred aspect, the oligonucleotideprimers represent at least part of the Nogo conserved segments of stronghomology between Nogo of different species. The syntheticoligonucleotides may be utilized as primers to amplify by PCR sequencesfrom a source (RNA or DNA), preferably a cDNA library, of potentialinterest. PCR can be carried out, e.g., by use of a Perkin-Elmer Cetusthermal cycler and Taq polymerase (Gene Amp™). The DNA being amplifiedcan include mRNA or cDNA or genomic DNA from any eukaryotic species. Onecan choose to synthesize several different degenerate primers, for usein the PCR reactions. It is also possible to vary the stringency ofhybridization conditions used in priming the PCR reactions, to allow forgreater or lesser degrees of nucleotide sequence similarity between theknown Nogo nucleotide sequence and the nucleic acid homolog beingisolated. For cross species hybridization, low stringency conditions arepreferred. For same species hybridization, moderately stringentconditions are preferred.

After successful amplification of a segment of a Nogo homolog, thatsegment may be molecularly cloned and sequenced, and utilized as a probeto isolate a complete cDNA or genomic clone. This, in turn, will permitthe determination of the gene's complete nucleotide sequence, theanalysis of its expression, and the production of its protein productfor functional analysis, as described infra. In this fashion, additionalgenes encoding Nogo proteins and Nogo analogs may be identified.

The above-methods are not meant to limit the following generaldescription of methods by which clones of Nogo may be obtained.

Any eukaryotic cell potentially can serve as the nucleic acid source forthe molecular cloning of the Nogo gene. The nucleic acid sequencesencoding Nogo can be isolated from vertebrate, mammalian, human,porcine, murine, bovine, feline, avian, equine, canine, as well asadditional primate sources, insects, etc. The DNA may be obtained bystandard procedures known in the art from cloned DNA (e.g., a DNA“library”), by chemical synthesis, by cDNA cloning, or by the cloning ofgenomic DNA, or fragments thereof, purified from the desired cell. (See,for example, Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.; Glover, D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRLPress, Ltd., Oxford, U.K. Vol. I, II.) Clones derived from genomic DNAmay contain regulatory and intron DNA regions in addition to codingregions; clones derived from cDNA will contain only exon sequences.Whatever the source, the gene should be molecularly cloned into asuitable vector for propagation of the gene.

In the molecular cloning of the gene from genomic DNA, DNA fragments aregenerated, some of which will encode the desired gene. The DNA may becleaved at specific sites using various restriction enzymes.Alternatively, one may use DNAse in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, as for example,by sonication. The linear DNA fragments can then be separated accordingto size by standard techniques, including but not limited to, agaroseand polyacrylamide gel electrophoresis and column chromatography.

Once the DNA fragments are generated, identification of the specific DNAfragment containing the desired gene may be accomplished in a number ofways. For example, if an amount of a portion of a Nogo (of any species)gene or its specific RNA, or a fragment thereof (see Section 6.1.1), isavailable and can be purified and labeled, the generated DNA fragmentsmay be screened by nucleic acid hybridization to the labeled probe(Benton, W. and Davis, R., 1977, Science 196:180; Grunstein, M. AndHogness, D., 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961). Those DNAfragments with substantial homology to the probe will hybridize. It isalso possible to identify the appropriate fragment by restriction enzymedigestion(s) and comparison of fragment sizes with those expectedaccording to a known restriction map if such is available. Furtherselection can be carried out on the basis of the properties of the gene.

Alternatively, the presence of the gene may be detected by assays basedon the physical, chemical, or immunological properties of its expressedproduct. For example, cDNA clones, or DNA clones which hybrid-select theproper mRNAs, can be selected which produce a protein that, e.g., hassimilar or identical electrophoretic migration, isolectric focusingbehavior, proteolytic digestion maps, post-translational modifications,acidic or basic properties or antigenic properties as known for Nogo.Antibodies to Nogo are available, such as IN-1 and IN-2 (U.S. Pat. No.5,684,133), AS Bruna and AS 472. Preparation of AS Bruna and AS 472 aredescribed in Section 6.1.7. The Nogo protein may be identified bybinding of labeled antibody to the putatively Nogo synthesizing clones,in an ELISA (enzyme-linked immunosorbent assay)-type procedure or bywestern blotting of purified or whole cell extracts.

The Nogo gene can also be identified by mRNA selection by nucleic acidhybridization followed by in vitro translation. In this procedure,fragments are used to isolate complementary mRNAs by hybridization. SuchDNA fragments may represent available, purified Nogo DNA of anotherspecies (e.g., mouse, human). Immunoprecipitation analysis or functionalassays (e.g., aggregation ability in vitro; binding to receptor; seeinfra) of the in vitro translation products of the isolated products ofthe isolated mRNAs identifies the mRNA and, therefore, the complementaryDNA fragments that contain the desired sequences. In addition, specificmRNAs may be selected by adsorption of polysomes isolated from cells toimmobilized antibodies specifically directed against Nogo protein. Aradiolabeled Nogo cDNA can be synthesized using the selected mRNA (fromthe adsorbed polysomes) as a template. The radiolabeled mRNA or cDNA maythen be used as a probe to identify the Nogo DNA fragments from amongother genomic DNA fragments.

Alternatives to isolating the Nogo genomic DNA include, but are notlimited to, chemically synthesizing the gene sequence itself from aknown sequence or making cDNA to the mRNA which encodes the Nogoprotein. For example, RNA for cDNA cloning of the Nogo gene can beisolated from cells which express Nogo. Other methods are possible andwithin the scope of the invention.

The identified and isolated gene can then be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art may be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Such vectors include, but are notlimited to, bacteriophages such as lambda derivatives, or plasmids suchas pBR322 or pUC plasmid derivatives or the Bluescript vector(Stratagene). In a specific example, Nogo is cloned into pcDNA3 withepitope tags for simplified protein expression analysis (Section6.1.10).

The insertion into a cloning vector can, for example, be accomplished byligating the DNA fragment into a cloning vector which has complementarycohesive termini. However, if the complementary restriction sites usedto fragment the DNA are not present in the cloning vector, the ends ofthe DNA molecules may be enzymatically modified. Alternatively, any sitedesired may be produced by ligating nucleotide sequences (linkers) ontothe DNA termini; these ligated linkers may comprise specific chemicallysynthesized oligonucleotides encoding restriction endonucleaserecognition sequences. In an alternative method, the cleaved vector andNogo gene may be modified by homopolymeric tailing. Recombinantmolecules can be introduced into host cells via transformation,transfection, infection, electroporation, etc., so that many copies ofthe gene sequence are generated.

In an alternative method, the desired gene may be identified andisolated after insertion into a suitable cloning vector in a “shot gun”approach. Enrichment for the desired gene, for example, by sizefractionization, can be done before insertion into the cloning vector.

In specific embodiments, transformation of host cells with recombinantDNA molecules that incorporate the isolated Nogo gene, cDNA, orsynthesized DNA sequence enables generation of multiple copies of thegene. Thus, the gene may be obtained in large quantities by growingtransformants, isolating the recombinant DNA molecules from thetransformants and, when necessary, retrieving the inserted gene from theisolated recombinant DNA.

The Nogo sequences provided by the instant invention include thosenucleotide sequences encoding substantially the same amino acidsequences as found in native Nogo proteins, and those encoded amino acidsequences with functionally equivalent amino acids, as well as thoseencoding other Nogo derivatives or analogs, as described in Sections6.2.1 and 6.2.2 infra for Nogo derivatives and analogs.

5.2 Expression of the Nogo Genes

The nucleotide sequence coding for a Nogo protein or a functionallyactive analog or fragment or other derivative thereof (see FIGS. 1 b and2 a; Sections 6.2.1 and 6.2.2), can be inserted into an appropriateexpression vector, i.e., a vector which contains the necessary elementsfor the transcription and translation of the inserted protein-codingsequence. The necessary transcriptional and translational signals canalso be supplied by the native Nogo gene and/or its flanking regions.The coding sequence can also be tagged with a sequence that codes for awell described antigen or biological molecule that has known bindingproperties to a binding partner (e.g. myc epitope tag, histidine tag, T7epitope tag etc., see Section 6.2.6 and FIG. 11 a-11 c). This additionalsequence can then be exploited to purify the Nogo protein, proteinfragment, or derivative using the interaction of the binding group withits corresponding partner, which is attached to a solid matrix.

A variety of host-vector systems may be utilized to express theprotein-coding sequence. These include but are not limited to mammaliancell systems infected with virus (e.g., vaccinia virus, adenovirus,etc.); insect cell systems infected with virus (e.g., baculovirus);microorganisms such as yeast containing yeast vectors, or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.In specific embodiments, the human Nogo gene is expressed, or a sequenceencoding a functionally active portion of human Nogo, as a specificexample, either Nogo A, Nogo B or Nogo C is expressed (FIG. 1 b). In yetanother embodiment, a fragment of Nogo comprising a domain of the Nogoprotein is expressed.

As used herein, a cell is “transformed” with a nucleic acid, when suchcell contains a nucleic acid not natively present in the cell, afterintroduction of the nucleic acid into the cell or its ancestor, e.g., bytransfection, electroporation, transduction, etc.

Nucleotide sequences encoding fragments of human Nogo A comprising anamino acid sequence selected from the group consisting of amino acidresidues 1-131, 132-939, 206-501, 501-680, 132-206, 680-939, and940-1127 of SEQ ID NO:29 are also provided. Nucleotide sequences thatencodes truncated portions of human Nogo A are also provided; thetruncated proteins lack amino acid residues 132-206, amino acid residues939-1127, or amino acid residues 132-206 and 939-1127, of SEQ ID NO:29but otherwise comprises the remainder of SEQ ID NO:29.

Any of the methods previously described for the insertion of DNAfragments into a vector may be used to construct expression vectorscontaining a chimeric gene consisting of appropriatetranscriptional/translational control signals and the protein codingsequences. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).Expression of nucleic acid sequence encoding a Nogo protein or peptidefragment may be regulated by a second nucleic acid sequence so that theNogo protein or peptide is expressed in a host transformed with therecombinant DNA molecule. For example, expression of a Nogo protein maybe controlled by any promoter/enhancer element known in the art. Anexemplary embodiment is to use one of Nogo's natural promoters, eitherP1 or P2, discussed in Section 6.2.1. A non-native promoter may also beused. Promoters which may be used to control Nogo expression include,but are not limited to, the SV40 early promoter region (Bernoist andChambon, 1981, Nature 290:304-310), the promoter contained in the 3′long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981,Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences ofthe metallothionein gene (Brinster et al., 1982, Nature 296:39-42);prokaryotic expression vectors such as the β-lactamase promoter(Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75:3727-3731), or the tac promoter (DeBoer, et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:21-25); see also “Useful proteins from recombinantbacteria” in Scientific American, 1980, 242:74-94; plant expressionvectors comprising the nopaline synthetase promoter region(Herrera-Estrella et al., Nature 303:209-213) or the cauliflower mosaicvirus 35S RNA promoter (Gardner, et al., 1981, Nucl. Acids Res. 9:2871),and the promoter of the photosynthetic enzyme ribulose biphosphatecarboxylase (Herrera-Estrella et al., 1984, Nature 310:115-120);promoter elements from yeast or other fungi such as the Gal 4 promoter,the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase)promoter, alkaline phosphatase promoter, and the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: elastase I gene control regionwhich is active in pancreatic acinar cells (Swift et al., 1984, Cell38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol.50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene controlregion which is active in pancreatic beta cells (Hanahan, 1985, Nature315:115-122), immunoglobulin gene control region which is active inlymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al.,1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol.7:1436-1444), mouse mammary tumor virus control region which is activein testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell45:485-495), albumin gene control region which is active in liver(Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoproteingene control region which is active in liver (Krumlauf et al., 1985,Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58;alpha 1-antitrypsin gene control region which is active in the liver(Kelsey et al., 1987, Genes and Devel. 1:161-171), beta-globin genecontrol region which is active in myeloid cells (Mogram et al., 1985,Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94; myelin basicprotein gene control region which is active in oligodendrocyte cells inthe brain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2gene control region which is active in skeletal muscle (Sani, 1985,Nature 314:283-286), and gonadotropic releasing hormone gene controlregion which is active in the hypothalamus (Mason et al., 1986, Science234:1372-1378).

In a specific embodiment, a vector is used that comprises a promoteroperably linked to a Nogo-encoding nucleic acid, one or more origins ofreplication, and, optionally, one or more selectable markers (e.g., anantibiotic resistance gene).

In a specific embodiment, an expression construct is made by subcloninga Nogo coding sequence into the EcoRI restriction site of each of thethree pGEX vectors (Glutathione S-Transferase expression vectors; Smithand Johnson, 1988, Gene 7:31-40). This allows for the expression of theNogo protein product from the subclone in the correct reading frame.

Expression vectors containing Nogo gene inserts can be identified bythree general approaches: (a) nucleic acid hybridization, (b) presenceor absence of “marker” gene functions, and (c) expression of insertedsequences. In the first approach, the presence of a Nogo gene insertedin an expression vector can be detected by nucleic acid hybridizationusing probes comprising sequences that are homologous to an insertedNogo gene. In the second approach, the recombinant vector/host systemcan be identified and selected based upon the presence or absence ofcertain “marker” gene functions (e.g., thymidine kinase activity,resistance to antibiotics, transformation phenotype, occlusion bodyformation in baculovirus, etc.) caused by the insertion of a Nogo genein the vector. For example, if the Nogo gene is inserted within themarker gene sequence of the vector, recombinants containing the Nogoinsert can be identified by the absence of the marker gene function. Inthe third approach, recombinant expression vectors can be identified byassaying the Nogo product expressed by the recombinant. Such assays canbe based, for example, on the physical or functional properties of theNogo protein in in vitro assay systems, e.g., binding with anti-Nogoantibody.

Once a particular recombinant DNA molecule is identified and isolated,several methods known in the art may be used to propagate it. Once asuitable host system and growth conditions are established, recombinantexpression vectors can be propagated and prepared in quantity. Aspreviously explained, the expression vectors which can be used include,but are not limited to, the following vectors or their derivatives:human or animal viruses such as vaccinia virus or adenovirus; insectviruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g.,lambda), and plasmid and cosmid DNA vectors, to name but a few.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thus,expression of the genetically engineered Nogo protein may be controlled.Furthermore, different host cells have characteristic and specificmechanisms for the translational and post-translational processing andmodification (e.g., glycosylation, phosphorylation of proteins).Appropriate cell lines or host systems can be chosen to ensure thedesired modification and processing of the foreign protein expressed.For example, expression in a bacterial system can be used to produce anunglycosylated core protein product. Expression in yeast will produce aglycosylated product. Expression in mammalian cells can be used toensure “native” glycosylation of a heterologous protein. Furthermore,different vector/host expression systems may effect processing reactionsto different extents.

In other specific embodiments, the Nogo protein, fragment, analog, orderivative may be expressed as a fusion, or chimeric protein product(comprising the protein, fragment, analog, or derivative joined via apeptide bond to a heterologous protein sequence (of a differentprotein)). Such a chimeric product can be made by ligating theappropriate nucleic acid sequences encoding the desired amino acidsequences to each other by methods known in the art, in the propercoding frame, and expressing the chimeric product by methods commonlyknown in the art. Alternatively, such a chimeric product may be made byprotein synthetic techniques, e.g., by use of a peptide synthesizer.

Both cDNA and genomic sequences can be cloned and expressed.

5.3 Identification and Purification of the Nogo Gene Products

In particular aspects, the invention provides amino acid sequences ofNogo, preferably human Nogo, and fragments and derivatives thereof whichcomprise an antigenic determinant (i.e., can be recognized by anantibody) or which are otherwise functionally active, as well as nucleicacid sequences encoding the foregoing. “Functionally active” Nogomaterial as used herein refers to that material displaying one or moreknown functional activities associated with a full-length (wild-type)Nogo A protein, e.g., non-permissive substrate properties, dorsal rootganglia growth cone collapse, NIH 3T3 spreading inhibition, neuriteoutgrowth inhibition, binding to a Nogo substrate or Nogo bindingpartner, antigenicity (binding to an anti-Nogo antibody),immunogenicity, etc.

In specific embodiments, the invention provides fragments of a Nogoprotein consisting of at least 6 amino acids, 10 amino acids, 17 aminoacids, 50 amino acids, 100 amino acids or of at least 220 amino acids.In other embodiments, the proteins comprise or consist essentially ofthe highly conserved Nogo carboxy terminal domain (carboxy terminal 188amino acids of Nogo A). Fragments, or proteins comprising fragments,lacking the conserved carboxy terminal domain, or the hydrophobiccarboxy terminal stretches, or the amino terminal acidic domain, or theamino terminal poly-proline region or any combination thereof, of a Nogoprotein are also provided. Nucleic acids encoding the foregoing areprovided.

Once a recombinant which expresses the Nogo gene sequence is identified,the gene product can be analyzed. This is achieved by assays based onthe physical or functional properties of the product, includingradioactive labeling of the product followed by analysis by gelelectrophoresis, immunoassay, etc.

Once the Nogo protein is identified, it may be isolated and purified bystandard methods including chromatography (e.g., ion exchange, affinity,and sizing column chromatography), centrifugation, differentialsolubility, or by any other standard technique for the purification ofproteins. The functional properties may be evaluated using any suitableassay including dorsal root ganglia growth cone collapse, NIH 3T3spreading inhibition, inhibition of neurite regeneration in optic nerves(see Sections 6.2.4-6.2.5).

Alternatively, once a Nogo protein produced by a recombinant isidentified, the amino acid sequence of the protein can be deduced fromthe nucleotide sequence of the chimeric gene contained in therecombinant. As a result, the protein can be synthesized by standardchemical methods known in the art (e.g., see Hunkapiller, M., et al.,1984, Nature 310:105-111).

In another alternate embodiment, native Nogo C, can be purified fromnatural sources, by standard methods such as those described above (e.g.immunoaffinity purification).

In a specific embodiment of the present invention, such Nogo proteins,whether produced by recombinant DNA techniques or by chemical syntheticmethods or by purification of native proteins, include but are notlimited to those containing, as a primary amino acid sequence, all orpart of the amino acid sequence substantially as depicted in FIG. 2 a(SEQ ID NO:2), bovine in FIG. 12 (SEQ ID NO:28), or human in FIG. 13(SEQ ID NO:29), as well as fragments and other derivatives (such as butnot limited to those depicted in FIG. 18), and analogs thereof,including proteins homologous thereto. Preferably, the Nogo proteins ofthe invention are free of all CNS myelin material with which it isnormally associated.

5.4 Structure of the Nogo Gene and Protein

The structure of the Nogo gene and protein can be analyzed by variousmethods known in the art and several of these methods are described inthe following subsections.

5.4.1 Genetic Analysis

The cloned DNA or cDNA corresponding to the Nogo gene can be analyzed bymethods including but not limited to Southern hybridization (Southern,E. M., 1975, J. Mol. Biol. 98:503-517), Northern hybridization (seee.g., Freeman et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:4094-4098),restriction endonuclease mapping (Maniatis, T., 1982, Molecular Cloning,A Laboratory, Cold Spring Harbor, N.Y.), and DNA sequence analysis.Polymerase chain reaction (PCR; U.S. Pat. Nos. 4,683,202, 4,683,195 and4,889,818; Gyllenstein et al., 1988, Proc. Natl. Acad. Sci. U.S.A.85:7652-7656; Ochman et al., 1988, Genetics 120:621-623; Loh et al.,1989, Science 243:217-220) followed by Southern hybridization with aNogo-specific probe can allow the detection of the Nogo gene in DNA fromvarious cell types. Methods of amplification other than PCR are commonlyknown and can also be employed. In one embodiment, Southernhybridization can be used to determine the genetic linkage of Nogo.Northern hybridization analysis can be used to determine the expressionof the Nogo gene. Various cell types, at various states of developmentor activity can be tested for Nogo expression. The stringency of thehybridization conditions for both Southern and Northern hybridizationcan be manipulated to ensure detection of nucleic acids with the desireddegree of relatedness to the specific Nogo probe used. Modifications ofthese methods and other methods commonly known in the art can be used.

Restriction endonuclease mapping can be used to roughly determine thegenetic structure of the Nogo gene. Restriction maps derived byrestriction endonuclease cleavage can be confirmed by DNA sequenceanalysis.

DNA sequence analysis can be performed by any techniques known in theart, including but not limited to the method of Maxam and Gilbert (1980,Meth. Enzymol. 65:499-560), the Sanger dideoxy method (Sanger, F., etal., 1977, Proc. Natl. Acad. Sci. U.S.A. 74:5463), the use of T7 DNApolymerase (Tabor and Richardson, U.S. Pat. No. 4,795,699), or use of anautomated DNA sequenator (e.g., Applied Biosystems, Foster City,Calif.).

5.4.2 Protein Analysis

The amino acid sequence of the Nogo protein can be derived by deductionfrom the DNA sequence, or alternatively, by direct sequencing of theprotein, e.g., with an automated amino acid sequencer.

The Nogo protein sequence can be further characterized by ahydrophilicity analysis (Hopp, T. and Woods, K., 1981, Proc. Natl. Acad.Sci. U.S.A. 78:3824). A hydrophilicity profile can be used to identifythe hydrophobic and hydrophilic regions of the Nogo protein and thecorresponding regions of the gene sequence which encode such regions.

Secondary, structural analysis (Chou, P. and Fasman, G., 1974,Biochemistry 13:222) can also be done, to identify regions of Nogo thatassume specific secondary structures.

Manipulation, translation, and secondary structure prediction, openreading frame prediction and plotting, as well as determination ofsequence homologies, can also be accomplished using computer softwareprograms available in the art.

Other methods of structural analysis can also be employed. These includebut are not limited to X-ray crystallography (Engstom, A., 1974,Biochem. Exp. Biol. 11:7-13) and computer modeling (Fletterick, R. andZoller, M. (eds.), 1986, Computer Graphics and Molecular Modeling, inCurrent Communications in Molecular Biology, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

5.5 Generation of Antibodies to Nogo Proteins and Derivatives Thereof

According to the invention, the Nogo protein, its fragments or otherderivatives, or analogs thereof, may be used as an immunogen to generateantibodies which immunospecifically bind such an immunogen. Suchantibodies include but are not limited to polyclonal, monoclonal,chimeric, single chain, Fab fragments, and an Fab expression library.Antibodies directed to a recombinant fragment of rat and bovine Nogo areproduced (Section 6.1.7), these antibodies also cross react with otherspecies epitopes. In another embodiment, fragments of a Nogo proteinidentified as hydrophilic are used as immunogens for antibodyproduction.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to a Nogo protein or derivative or analog. In aparticular embodiment, rabbit polyclonal antibodies to an epitope of aNogo protein encoded by a sequence of SEQ ID NO:2 in FIG. 2 a, SEQ IDNO:28 in FIG. 12, SEQ ID NO:32 in FIG. 14, or SEQ ID NO:29 in FIG. 13,(rat Nogo A, bovine Nogo, rat Nogo C, or human Nogo respectively) or asubsequence thereof, can be obtained. For the production of antibody,various host animals can be immunized by injection with the native Nogoprotein, or a synthetic version, or derivative (e.g., fragment) thereof,including but not limited to rabbits, mice, rats, etc. Various adjuvantsmay be used to increase the immunological response, depending on thehost species, and including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanins, dinitrophenol, andpotentially useful human adjuvants such as BCG (bacille Calmette-Guerin)and corynebacterium parvum.

For preparation of monoclonal antibodies directed toward a Nogo proteinsequence or analog thereof, any technique which provides for theproduction of antibody molecules by continuous cell lines in culture maybe used. For example, the hybridoma technique originally developed byKohler and Milstein (1975, Nature 256:495-497), as well as the triomatechnique, the human B-cell hybridoma technique (Kozbor et al., 1983,Immunology Today 4:72), and the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96). Monoclonal antibodiescan be produced in germ-free animals utilizing recent technology(PCT/US90/02545). According to the invention, human antibodies may beused and can be obtained by using human hybridomas (Cote et al., 1983,Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030). or by transforming human Bcells with EBV virus in vitro (Cole et al., 1985, in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, pp. 77-96). In fact,according to the invention, techniques developed for the production of“chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.U.S.A. 81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takedaet al., 1985, Nature 314:452-454) by splicing the genes from a mouseantibody molecule specific for Nogo together with genes from a humanantibody molecule of appropriate biological activity can be used; suchantibodies are within the scope of this invention.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce Nogo-specific single chain antibodies. Techniques described forthe construction of Fab expression libraries can also be utilized (Huseet al., 1989, Science 246:1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificityfor Nogo proteins, derivatives, or analogs.

Antibody fragments which contain the idiotype of the molecule can begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab′)₂ fragment which can be produced by pepsindigestion of the antibody molecule; the Fab′ fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragment, theFab fragments which can be generated by treating the antibody moleculewith papain and a reducing agent, and Fv fragments.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g. ELISA(enzyme-linked immunosorbent assay). For example, to select antibodieswhich recognize a specific domain of a Nogo protein, one may assaygenerated hybridomas for a product which binds to a Nogo fragmentcontaining such domain. For selection of an antibody that specificallybinds a first Nogo homolog but which does not specifically bind adifferent Nogo homolog, one can select on the basis of positive bindingto the first Nogo homolog and a lack of binding to the second Nogohomolog.

Antibodies specific to a domain of a Nogo protein are also provided.

The foregoing antibodies can be used in methods known in the artrelating to the localization and activity of the Nogo protein sequencesof the invention, e.g., for imaging these proteins, measuring levelsthereof in appropriate physiological samples, in diagnostic methods,etc.

Anti-Nogo antibodies and fragments thereof containing the binding domainare Therapeutics.

5.6 Nogo Proteins, Derivatives and Analogs

The invention further relates to Nogo proteins, and derivatives(including but not limited to fragments) and analogs of Nogo proteins.Nucleic acids encoding Nogo protein derivatives and protein analogs arealso provided. In one embodiment, the Nogo proteins are encoded by theNogo nucleic acids described in Section 5.1 supra. In particularaspects, Nogo A, Nogo B, or Nogo C proteins and derivatives, or analogsare of animals, e.g., mouse, rat, pig, cow, dog, monkey, human, fly, orfrogs are within the scope of the invention.

The production and use of derivatives and analogs related to Nogo arealso within the scope of the present invention. In a specificembodiment, the derivative or analog is functionally active, i.e.,capable of exhibiting one or more functional activities associated witha full-length, wild-type Nogo protein. As one example, such derivativesor analogs which have the desired immunogenicity or antigenicity can beused, for example, in immunoassays, for immunization, for inhibition ofNogo activity, etc. Derivatives or analogs that retain, or alternativelylack or inhibit, a desired Nogo property of interest (e.g., binding to aNogo binding partner, can be used as inducers, or inhibitors,respectively, of such property and its physiological correlates. Aspecific embodiment relates to a Nogo fragment that can be bound by ananti-Nogo antibody. Derivatives or analogs of Nogo can be tested for thedesired activity by procedures known in the art, including but notlimited to the assays described in Sections 6.1.10 to 6.1.12.

In order to map the active region(s) of Nogo, a series of Nogo deletionmutants have been prepared by recombinant DNA techniques as described inSection 6.2.7. The portions of Nogo which are present in the deletionmutants are shown in FIG. 18. In a specific embodiment, the inventionprovides fragments of Nogo e.g., fragments comprising (or alternativelyconsisting of) Nogo A (SEQ ID NO: 2) amino acid numbers 1-171, 172-974,259-542, 542-722, 722-974, 172-259, or 975-1162, or combinations of theforegoing. Truncated mutants of Nogo lacking amino acid numbers 172-259and/or 975-1162 of SEQ ID NO:2 are also provided, as these regionsappear to be non-essential and can be removed from Nogo withoutaffecting biological activity. The corresponding fragments of human NogoA comprising (or alternatively consisting of) amino acid numbers 1-131,132-939, 206-501, 501-680, 132-206, 680-939, or 940-1127 of SEQ ID NO:29are also provided. Truncated mutants of human Nogo A are also providedwhich lack amino acid numbers 132-206, amino acid residues 939-1127, oramino acid residues 132-206 and 939-1127, of SEQ ID NO:29.

In a specific embodiment, the fragments are free of all CNS myelinmaterial and/or display inhibitory activity of Nogo. Fusion proteinscomprising one or more of the above fragments fused to a non-Nogosequence are also provided.

Nogo gene derivatives can be made by altering Nogo sequences bysubstitutions, additions or deletions that provide for functionallyequivalent molecules. Due to the degeneracy of nucleotide codingsequences, other DNA sequences which encode substantially the same aminoacid sequence as a Nogo gene may be used in the practice of the presentinvention. These include but are not limited to nucleotide sequencescomprising all or portions of Nogo genes which are altered by thesubstitution of different codons that encode a functionally equivalentamino acid residue within the sequence, thus producing a silent change.Likewise, the Nogo derivatives of the invention include, but are notlimited to, those containing, as a primary amino acid sequence, all orpart of the amino acid sequence of a Nogo protein including alteredsequences in which functionally equivalent amino acid residues aresubstituted for residues within the sequence resulting in a silentchange. For example, one or more amino acid residues within the sequencecan be conservatively substituted by another amino acid of a similarpolarity which acts as a functional equivalent, resulting in a silentalteration. Substitutes for an amino acid within the sequence may beselected from other members of the class to which the amino acidbelongs. For example, the nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophanand methionine. The polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine. The positivelycharged (basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid.

In a specific embodiment of the invention, proteins consisting of orcomprising a fragment of a Nogo protein consisting of at least 10(continuous) amino acids of the Nogo protein is provided. In otherembodiments, the fragment consists of at least 17 or 50 amino acids ofthe Nogo protein. In specific embodiments, such fragments are not largerthan 35, 100 or 200 amino acids. Derivatives or analogs of Nogo includebut are not limited to those molecules comprising regions that aresubstantially homologous to Nogo or fragments thereof (e.g., in variousembodiments, at least 60% or 70% or 80% or 90% or 95% identity over anamino acid sequence of identical size or when compared to an alignedsequence in which the alignment is done by a computer homology programknown in the art, for example BLAST computer searching (Altschul et al.,1994, Nature Genet. 6:119-129)) or whose encoding nucleic acid iscapable of hybridizing to a coding Nogo sequence, under stringent,moderately stringent, or nonstringent conditions.

Molecules comprising Nogo fragments are also provided, e.g., containinghydrocarbon linkages to other moieties including labels or bioactivemoieties.

The Nogo derivatives and analogs of the invention can be produced byvarious methods known in the art. The manipulations which result intheir production can occur at the gene or protein level. For example,the cloned Nogo gene sequence can be modified by any of numerousstrategies known in the art (Maniatis, T., 1990, Molecular Cloning, ALaboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.). The sequence can be cleaved at appropriate sites withrestriction endonuclease(s), followed by further enzymatic modificationif desired, isolated, and ligated in vitro. In the production of thegene encoding a derivative or analog of Nogo, care should be taken toensure that the modified gene remains within the same translationalreading frame as Nogo, uninterrupted by translational stop signals, inthe gene region where the desired Nogo activity is encoded.

Additionally, the Nogo-encoding nucleic acid sequence can be mutated invitro or in vivo, to create and/or destroy translation, initiation,and/or termination sequences, or to create variations in coding regionsand/or form new restriction endonuclease sites or destroy preexistingones, to facilitate further in vitro modification. Any technique formutagenesis known in the art can be used, including but not limited to,chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson,C., et al., 1978, J. Biol. Chem 253:6551), use of TAB® linkers(Pharmacia), etc.

Manipulations of the Nogo sequence may also be made at the proteinlevel. Included within the scope of the invention are Nogo proteinfragments or other derivatives or analogs which are differentiallymodified during or after translation, e.g., by glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligand, etc. Any of numerous chemicalmodifications may be carried out by known techniques, including but notlimited to specific chemical cleavage by cyanogen bromide, trypsin,chymotrypsin, papain, V8 protease, NaBH₄; acetylation, formylation,oxidation, reduction; metabolic synthesis in the presence oftunicamycin; etc.

In addition, analogs and derivatives of Nogo can be chemicallysynthesized. For example, a peptide corresponding to a portion of a Nogoprotein which comprises the desired domain or which mediates the desiredactivity in vitro, can be synthesized by use of a peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into the Nogosequence. Non-classical amino acids include but are not limited to theD-isomers of the common amino acids, α-amino isobutyric acid,4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid,ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acidssuch as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

In a specific embodiment, the Nogo derivative is a chimeric, or fusion,protein comprising a Nogo protein or fragment thereof (preferablyconsisting of at least a domain or motif of the Nogo protein, or atleast 10 amino acids of the Nogo protein) joined at its amino- orcarboxy-terminus via a peptide bond to an amino acid sequence of adifferent protein. In one embodiment, such a chimeric protein isproduced by recombinant expression of a nucleic acid encoding theprotein (comprising a Nogo-coding sequence joined in-frame to a codingsequence for a different protein). Such a chimeric product can be madeby ligating the appropriate nucleic acid sequences encoding the desiredamino acid sequences to each other by methods known in the art, in theproper coding frame, and expressing the chimeric product by methodscommonly known in the art. Alternatively, such a chimeric product may bemade by protein synthetic techniques, e.g., by use of a peptidesynthesizer. Chimeric genes comprising portions of Nogo fused to anyheterologous protein-encoding sequences may be constructed. Suchheterologous protein-encoding sequences include, for example, thehexahistidine tag, and the T7 tag. A specific embodiment relates to achimeric protein comprising a fragment of Nogo of at least six aminoacids.

In another specific embodiment, the Nogo derivative is a moleculecomprising a region of homology with a Nogo protein.

In a preferred embodiment, the Nogo derivatives (e.g., fragments) areproteins that are non-naturally occurring.

Other specific embodiments of derivatives and analogs are described inthe subsection below and examples sections infra.

5.6.1 Derivatives of Nogo Containing One or More Domains of the Protein

In a specific embodiment, the invention relates to Nogo derivatives andanalogs, in particular Nogo fragments and derivatives of such fragments,that comprise, or alternatively consist of, one or more domains of aNogo protein, including but not limited to the conserved carboxyterminal and hydrophobic domains or the amino terminal acidic or polyproline rich domains, functional (e.g., binding) fragments of any of theforegoing, or any combination of the foregoing.

A specific embodiment relates to molecules comprising specific fragmentsof Nogo that are those fragments in the respective Nogo protein mosthomologous to specific fragments of a rat or bovine Nogo protein. Afragment comprising a domain of a Nogo homolog can be identified byprotein analysis methods as described in Sections 6.1.2, 6.1.8, 6.1.9,6.1.10, 6.1.11, or 6.1.12.

In another specific embodiment, a molecule is provided that comprisesone or more domains (or functional portion thereof) of a Nogo proteinbut that also lacks one or more domains (or functional portion thereof)of a Nogo protein. In another embodiment, a molecule is provided thatcomprises one or more domains (or functional portion thereof) of a Nogoprotein, and that has one or more mutant (e.g., due to deletion or pointmutation(s)) domains of a Nogo protein (e.g., such that the mutantdomain has decreased function).

5.7 Assays of Nogo Proteins, Derivatives and Analogs

The functional activity of Nogo proteins, derivatives and analogs can beassayed by various methods. The description of functional assays in thefollowing sections are not meant to be limiting, and may include otherassays known to one skilled in the art.

5.7.1 Assays of Nogo In Vitro Neurite Growth Inhibition

In a specific embodiment, Nogo proteins, derivatives and analogs can beassayed for inhibition of NIH 3T3 spreading or inhibition of PC12neurite outgrowth using in vitro tissue culture (Section 6.1.10).

In an alternative embodiment, Nogo proteins, derivatives and analogs canbe used to assay for explanted chick dorsal root ganglia growth conecollapse induced by the presence of Nogo. Similarly, Nogo function canbe assayed for inhibition of neurite outgrowth of explanted chick dorsalroot ganglia (Spillman et al., 1998 J. Biol. Chem. 273:19283-19293).

5.7.2 Assays of Nogo In Vivo Functional Properties

In one example, antagonists of Nogo proteins, derivatives and analogscan be used for in vivo assays of function using an animal model forcorticospinal tract (CST) regeneration over long distances and behaviorrecovery.

In a preferred embodiment, a rodent corticospinal tract is damaged bysurgical resection or spinal cord contusion, and antagonists of Nogo areadministered to the animal. Neural plasticity, regeneration andfunctional recovery, as compared to untreated control animals or controlantibody treated animals, are monitored for structural plasticity orregeneration by anatomical techniques, mainly by labeling of definedneural tracts. Functional recovery is measured by locomotion and byelectrophysiology skill tests executed by the rodent (e.g. sticky papertest, food pellet reaching task, etc.) (Thallmair et al., 1998 Nat.Neuroscience 1(2):124-131).

5.7.3 Nogo Ligand Binding Inhibition and Assays Thereof

In one embodiment, where one is assaying for the ability to bind orcompete with wild-type Nogo for binding to anti-Nogo antibody, variousimmunoassays known in the art can be used, including but not limited tocompetitive and non-competitive assay systems using techniques such asradioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitinreactions, immunodiffusion assays, in situ immunoassays (using colloidalgold, enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention.

In another embodiment, where a Nogo-binding protein is identified, thebinding can be assayed, e.g., by means well-known in the art. In anotherembodiment, physiological correlates of Nogo binding to its substratescan be assayed.

Other methods will be known to the skilled artisan and are within thescope of the invention.

5.8 Therapeutic Uses

The invention provides for treatment or prevention of various diseasesand disorders by administration of a therapeutic compound (termed herein“Therapeutic”). Such “Therapeutics” include but are not limited to: Nogoproteins and analogs and derivatives (including fragments) thereof(e.g., as described hereinabove); antibodies thereto (as describedhereinabove); nucleic acids encoding the Nogo proteins, analogs, orderivatives (e.g., as described hereinabove); Nogo antisense nucleicacids, and Nogo agonists and antagonists. Disorders involvingderegulated cellular growth, e.g. CNS tumors, are treated or preventedby administration of a Therapeutic that promotes Nogo function.Disorders in which neurite growth, regeneration, or maintenance aredeficient or desired are treated by administration of a Therapeutic thatantagonizes (inhibits) Nogo function. The above is described in detailin the subsections below.

Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, a humanNogo protein, derivative, or analog, or nucleic acid, or an antibody toa human Nogo protein, is therapeutically or prophylacticallyadministered to a human patient.

5.8.1 Treatment and Prevention of Disorders Involving DeregulatedCellular Growth

Diseases and disorders involving deregulated cellular growth are treatedor prevented by administration of a Therapeutic that promotes (i.e.,increases or supplies) Nogo function. Examples of such a Therapeuticinclude but are not limited to Nogo proteins, derivatives, or fragmentsthat are functionally active, particularly that are active in inhibitionof neurite extension or cellular growth inhibition (e.g., asdemonstrated in in vitro assays or in animal models), and nucleic acidsencoding a Nogo protein or functionally active derivative or fragmentthereof (e.g., for use in gene therapy). Preferably, the Nogo proteins,derivatives or fragments thereof are free of all CNS myelin materialwith which it is naturally associated. Other Therapeutics that can beused, e.g., Nogo agonists, can be identified using in vitro assays oranimal models, examples of which are described infra.

In specific embodiments, Therapeutics that promote Nogo function areadministered therapeutically (including prophylactically): (1) indiseases or disorders involving an absence or decreased (relative tonormal or desired) level of Nogo protein or function, for example, inpatients where Nogo protein is lacking, genetically defective,biologically inactive or underactive, or underexpressed; or (2) indiseases or disorders wherein in vitro (or in vivo) assays (see infra)indicate the utility of Nogo agonist administration. The absence ordecreased level in Nogo protein or function can be readily detected,e.g., by obtaining a patient tissue sample (e.g., from biopsy tissue)and assaying it in vitro for RNA or protein levels, structure and/oractivity of the expressed Nogo RNA or protein. Many methods standard inthe art can be thus employed, including but not limited to kinaseassays, immunoassays to detect and/or visualize Nogo protein (e.g.,Western blot, immunoprecipitation followed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/orhybridization assays to detect Nogo expression by detecting and/orvisualizing Nogo mRNA (e.g., Northern assays, dot blots, in situhybridization, etc.), etc.

Diseases and disorders involving deregulated cellular growth that can betreated or prevented include but are not limited to proliferativedisorders, malignant tumors, nervous system tumors, etc. Examples ofthese are detailed below.

5.8.1.1 Neoplastic Growth

Neoplastic growth and related disorders that can be treated or preventedby administration of a Therapeutic that promotes Nogo function includebut are not limited to those listed in Table 1 (for a review of suchdisorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. LippincottCo., Philadelphia):

TABLE 1 NEOPLASTIC GROWTH AND RELATED DISORDERS Solid tumors sarcomasand carcinomas glioma, glioblastoma astrocytoma medulloblastomacraniopharyngioma ependymoma pinealoma hemangioblastoma acoustic neuromaoligodendroglioma menangioma neuroblastoma retinoblastoma

In specific embodiments, malignancy or dysproliferative changes (such asmetaplasias and dysplasias), or hyperproliferative disorders, aretreated or prevented in the central nervous system, spinal cord or anyneural tissues.

5.8.1.2 Premalignant Conditions

The Therapeutics of the invention that promote Nogo activity can also beadministered to treat premalignant conditions and to prevent progressionto a neoplastic or malignant state, including but not limited to thosedisorders listed in Table 1. Such prophylactic or therapeutic use isindicated in conditions known or suspected of preceding progression toneoplasia or cancer, in particular, where non-neoplastic cell growthconsisting of hyperplasia, metaplasia, or most particularly, dysplasiahas occurred (for review of such abnormal growth conditions, see Robbinsand Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co.,Philadelphia, pp. 68-79.) Hyperplasia is a form of controlled cellproliferation involving an increase in cell number in a tissue or organ,without significant alteration in structure or function. Metaplasia is aform of controlled cell growth in which one type of adult or fullydifferentiated cell substitutes for another type of adult cell.Metaplasia can occur in epithelial or connective tissue cells. Atypicalmetaplasia involves a somewhat disorderly metaplastic epithelium.Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia; it is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplastic cells often haveabnormally large, deeply stained nuclei, and exhibit pleomorphism.Dysplasia characteristically occurs where there exists chronicirritation or inflammation.

Alternatively or in addition to the presence of abnormal cell growthcharacterized as hyperplasia, metaplasia, or dysplasia, the presence ofone or more characteristics of a transformed phenotype, or of amalignant phenotype, displayed in vivo or displayed in vitro by a cellsample from a patient, can indicate the desirability ofprophylactic/therapeutic administration of a Therapeutic that promotesNogo function. As mentioned supra, such characteristics of a transformedphenotype include morphology changes, looser substratum attachment, lossof contact inhibition, loss of anchorage dependence, protease release,increased sugar transport, decreased serum requirement, expression offetal antigens, disappearance of the 250,000 dalton cell surfaceprotein, etc. (see also id., at pp. 84-90 for characteristics associatedwith a transformed or malignant phenotype).

In other embodiments, a patient which exhibits one or more of thefollowing predisposing factors for malignancy is treated byadministration of an effective amount of a Therapeutic:neurofibromatosis of Von Recklinghausen or retinoblastoma; see Robbinsand Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co.,Philadelphia, pp. 112-113) etc.)

In another specific embodiment, a Therapeutic of the invention isadministered to a human patient to prevent progression to kidney,cartilage (of the breast bone), skin, skeletal muscle, lung, or spleenof cancer, melanoma, or sarcoma.

5.8.1.3 Hyperproliferative and Dysproliferative Disorders

In another embodiment of the invention, a Therapeutic that promotes Nogoactivity is used to treat or prevent hyperproliferative or benigndysproliferative disorders. Specific embodiments are directed totreatment or prevention of cirrhosis of the liver (a condition in whichscarring has overtaken normal liver regeneration processes), treatmentof keloid (hypertrophic scar) formation (disfiguring of the skin inwhich the scarring process interferes with normal renewal), psoriasis (acommon skin condition characterized by excessive proliferation of theskin and delay in proper cell fate determination), benign tumors,fibrocystic conditions, and tissue hypertrophy (e.g., prostatichyperplasia).

5.8.1.4 Gene Therapy

In a specific embodiment, nucleic acids comprising a sequence encoding aNogo protein or functional derivative thereof, are administered topromote Nogo function, by way of gene therapy. Gene therapy refers totherapy performed by the administration of a nucleic acid to a subject.In this embodiment of the invention, the nucleic acid produces itsencoded protein that mediates a therapeutic effect by promoting Nogofunction.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann.Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11 (5):155-215). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), 1993, Current Protocols inMolecular Biology, John Wiley & Sons, NY; and Kriegler, 1990, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY.

In a preferred aspect, the Therapeutic comprises a Nogo nucleic acidthat is part of an expression vector that expresses a Nogo protein orfragment or chimeric protein thereof in a suitable host. In particular,such a nucleic acid has a promoter operably linked to the Nogo codingregion, said promoter being inducible or constitutive, and, optionally,tissue-specific. In another particular embodiment, a nucleic acidmolecule is used in which the Nogo coding sequences and any otherdesired sequences are flanked by regions that promote homologousrecombination at a desired site in the genome, thus providing forintrachromosomal expression of the Nogo nucleic acid (Koller andSmithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra etal., 1989, Nature 342:435-438).

Delivery of the nucleic acid into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vector, or indirect, in which case, cells arefirst transformed with the nucleic acid in vitro, then transplanted intothe patient. These two approaches are known, respectively, as in vivo orex vivo gene therapy.

In a specific embodiment, the nucleic acid is directly administered invivo, where it is expressed to produce the encoded product. This can beaccomplished by any of numerous methods known in the art, e.g., byconstructing it as part of an appropriate nucleic acid expression vectorand administering it so that it becomes intracellular, e.g., byinfection using a defective or attenuated retroviral or other viralvector (see U.S. Pat. No. 4,980,286), or by direct injection of nakedDNA, or by use of microparticle bombardment (e.g., a gene gun;Biolistic, Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, encapsulation in liposomes, microparticles, ormicrocapsules, or by administering it in linkage to a peptide which isknown to enter the nucleus, by administering it in linkage to a ligandsubject to receptor-mediated endocytosis (see e.g., Wu and Wu, 1987, J.Biol. Chem. 262:4429-4432) (which can be used to target cell typesspecifically expressing the receptors), etc. In another embodiment, anucleic acid-ligand complex can be formed in which the ligand comprisesa fusogenic viral peptide to disrupt endosomes, allowing the nucleicacid to avoid lysosomal degradation. In yet another embodiment, thenucleic acid can be targeted in vivo for cell specific uptake andexpression, by targeting a specific receptor (see, e.g., PCTPublications WO 92/06180 dated Apr. 16, 1992 (Wu et al.); WO 92/22635dated Dec. 23, 1992 (Wilson et al.); WO92/20316 dated Nov. 26, 1992(Findeis et al.); WO93/14188 dated Jul. 22, 1993 (Clarke et al.), WO93/20221 dated Oct. 14, 1993 (Young)). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, 1989,Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature342:435-438).

In a specific embodiment, a viral vector that contains the Nogo nucleicacid is used. For example, a retroviral vector can be used (see Milleret al., 1993, Meth. Enzymol. 217:581-599). These retroviral vectors havebeen modified to delete retroviral sequences that are not necessary forpackaging of the viral genome and integration into host cell DNA. TheNogo nucleic acid to be used in gene therapy is cloned into the vector,which facilitates delivery of the gene into a patient. More detail aboutretroviral vectors can be found in Boesen et al., 1994, Biotherapy6:291-302, which describes the use of a retroviral vector to deliver themdr1 gene to hematopoietic stem cells in order to make the stem cellsmore resistant to chemotherapy. Other references illustrating the use ofretroviral vectors in gene therapy are: Clowes et al., 1994, J. Clin.Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons andGunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson,1993, Curr. Opin. in Genetics and Devel. 3:110-114.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes tothe central nervous system. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the respiratory epithelia,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, 1993,Current Opinion in Genetics and Development 3:499-503 present a reviewof adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy5:3-10 demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al., 1991,Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155; andMastrangeli et al., 1993, J. Clin. Invest. 91:225-234.

In addition to Adenoviruses, Adeno-associated virus (AAV) has also beenproposed for use in gene therapy (Walsh et al., 1993, Proc. Soc. Exp.Biol. Med. 204:289-300.

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see e.g., Loeffler and Behr, 1993, Meth.Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644;Cline, 1985, Pharmac. Ther. 29:69-92) and may be used in accordance withthe present invention, provided that the necessary developmental andphysiological functions of the recipient cells are not disrupted. Thetechnique should provide for the stable transfer of the nucleic acid tothe cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. In a preferred embodiment, epithelial cellsare injected, e.g., subcutaneously. In another embodiment, recombinantskin cells may be applied as a skin graft onto the patient. Recombinantblood cells (e.g., hematopoietic stem or progenitor cells) arepreferably administered intravenously. The amount of cells envisionedfor use depends on the desired effect, patient state, etc., and can bedetermined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy, aNogo nucleic acid is introduced into the cells such that it isexpressible by the cells or their progeny, and the recombinant cells arethen administered in vivo for therapeutic effect. In a specificembodiment, stem or progenitor cells are used. Any stem and/orprogenitor cells which can be isolated and maintained in vitro canpotentially be used in accordance with this embodiment of the presentinvention. Such stem cells include but are not limited to neural stemcells (Stemple and Anderson, 1992, Cell 71:973-985).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

Additional methods that can be adapted for use to deliver a nucleic acidencoding a Nogo protein or functional derivative.

5.8.2 Treatment and Prevention of Disorders in which Nogo BlocksRegeneration

Diseases and disorders in which neurite extension, growth orregeneration are desired are treated by administration of a Therapeuticthat antagonizes (inhibits) Nogo function. The diseases, disorders ordamage which ultimately result in damage of the nervous system include,but are not limited to, central nervous system (CNS) trauma, (e.g.spinal cord or brain injuries), infarction, infection, malignancy,exposure to toxic agents, nutritional deficiency, paraneoplasticsyndromes, and degenerative nerve diseases (including but not limited toAlzheimer's disease, Parkinson's disease, Huntington's Chorea, multiplesclerosis, amyotrophic lateral sclerosis, and progressive supra-nuclearpalsy); by administering compounds that interfere with Nogo activity(e.g., a dominant negative Nogo derivative; antibodies to Nogo;anti-sense nucleic acids that encode Nogo; Nogo ribozymes or chemicalgroups that bind an active site of Nogo).

Therapeutics that can be used include but are not limited to Nogoantisense nucleic acids, and Nogo nucleic acids that are dysfunctional(e.g., due to a heterologous (non-Nogo sequence) insertion within theNogo coding sequence) that are used to “knockout” endogenous Nogofunction by homologous recombination (see, e.g., Capecchi, 1989, Science244:1288-1292). Anti-Nogo antibodies (and fragments and derivativesthereof containing the binding region thereof) can be used as anantagonist of Nogo. In a specific embodiment of the invention, a nucleicacid containing a portion of a Nogo gene in which Nogo sequences flank(are both 5′ and 3′ to) a different gene sequence, is used, as a Nogoantagonist, to promote Nogo inactivation by homologous recombination(see also Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438). OtherTherapeutics that inhibit Nogo function can be identified by use ofknown convenient in vitro assays, e.g., based on their ability toinhibit binding of Nogo to another protein, or inhibit any known Nogofunction, as preferably assayed in vitro or in cell culture, althoughgenetic assays may also be employed. Preferably, suitable in vitro or invivo assays, are utilized to determine the effect of a specificTherapeutic and whether its administration is indicated for treatment ofthe affected tissue.

In specific embodiments, Therapeutics that inhibit Nogo function areadministered therapeutically (including prophylactically): (1) indiseases or disorders involving an increased (relative to normal ordesired) level of Nogo protein or function, for example, in patientswhere Nogo protein is overactive or overexpressed; or (2) in diseases ordisorders wherein in vitro (or in vivo) assays (see infra) indicate theutility of Nogo antagonist administration. The increased levels in Nogoprotein or function can be readily detected, e.g., by quantifyingprotein and/or RNA, by obtaining a patient tissue sample (e.g., frombiopsy tissue) and assaying it in vitro for RNA or protein levels,structure and/or activity of the expressed Nogo RNA or protein. Manymethods standard in the art can be thus employed, including but notlimited to kinase assays, immunoassays to detect and/or visualize Nogoprotein (e.g., Western blot, immunoprecipitation followed by sodiumdodecyl sulfate polyacrylamide gel electrophoresis, immunocytochemistry,etc.) and/or hybridization assays to detect Nogo expression by detectingand/or visualizing respectively Nogo mRNA (e.g., Northern assays, dotblots, in situ hybridization, etc.), etc.

5.8.2.1 Antisense Regulation of Nogo Expression

In a specific embodiment, Nogo function is inhibited by use of Nogoantisense nucleic acids. The present invention provides the therapeuticor prophylactic use of nucleic acids of at least six nucleotides thatare antisense to a gene or cDNA encoding Nogo or a portion thereof. ANogo “antisense” nucleic acid as used herein refers to a nucleic acidcapable of hybridizing to a portion of a Nogo RNA (preferably mRNA) byvirtue of some sequence complementarity. The antisense nucleic acid maybe complementary to a coding and/or noncoding region of a Nogo mRNA.Such antisense nucleic acids have utility as Therapeutics that inhibitNogo function, and can be used in the treatment or prevention ofdisorders as described supra.

The antisense nucleic acids of the invention can be oligonucleotidesthat are double-stranded or single-stranded, RNA or DNA or amodification or derivative thereof, which can be directly administeredto a cell, or which can be produced intracellularly by transcription ofexogenous, introduced sequences.

In a specific embodiment, the Nogo antisense nucleic acids provided bythe instant invention can be used to promote regeneration of neurons ofthe central nervous system in particular, including regeneration of thecorticospinal tract, plasticity during recovery, regrowth of neurons andhealing of damage associated with traumatic injuries, strokes, andneurodegenerative diseases.

The invention further provides pharmaceutical compositions comprising aneffective amount of the Nogo antisense nucleic acids of the invention ina pharmaceutically acceptable carrier, as described infra.

In another embodiment, the invention is directed to methods forinhibiting the expression of a Nogo nucleic acid sequence in aprokaryotic or eukaryotic cell comprising providing the cell with aneffective amount of a composition comprising an Nogo antisense nucleicacid of the invention.

Nogo antisense nucleic acids and their uses are described in detailbelow.

5.8.2.1.1 Nogo Antisense Nucleic Acids

The Nogo antisense nucleic acids are of at least six nucleotides and arepreferably oligonucleotides (ranging from 6 to about 50oligonucleotides). In specific aspects, the oligonucleotide is at least10 nucleotides, at least 15 nucleotides, at least 100 nucleotides, or atleast 200 nucleotides. The oligonucleotides can be DNA or RNA orchimeric mixtures or derivatives or modified versions thereof,single-stranded or double-stranded. The oligonucleotide can be modifiedat the base moiety, sugar moiety, or phosphate backbone. Theoligonucleotide may include other appending groups such as peptides, oragents facilitating transport across the cell membrane (see, e.g.,Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556;Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCTPublication No. WO 88/09810, published Dec. 15, 1988) or blood-brainbarrier (see, e.g., PCT Publication No. WO 89/10134, published Apr. 25,1988), hybridization-triggered cleavage agents (see, e.g., Krol et al.,1988, BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon,1988, Pharm. Res. 5:539-549).

In a preferred aspect of the invention, a Nogo antisense oligonucleotideis provided, preferably of single-stranded DNA. In a most preferredaspect, such an oligonucleotide comprises a sequence antisense to thesequence near one of the two promoter sequences of the Nogo gene, or asequence encoding carboxy-terminal portion of the Nogo gene. It may bedesirable to selectively inhibit the expression one of the Nogoisoforms. The oligonucleotide may be modified at any position on itsstructure with substituents generally known in the art.

The Nogo antisense oligonucleotide may comprise at least one modifiedbase moiety which is selected from the group including but not limitedto 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

In another embodiment, the oligonucleotide comprises at least onemodified sugar moiety selected from the group including but not limitedto arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the oligonucleotide comprises at least onemodified phosphate backbone selected from the group consisting of aphosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the oligonucleotide is an α-anomericoligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641).

The oligonucleotide may be conjugated to another molecule, e.g., apeptide, hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

Oligonucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

In a specific embodiment, the Nogo antisense oligonucleotide comprisescatalytic RNA, or a ribozyme (see, e.g., PCT International PublicationWO 90/11364, published Oct. 4, 1990; Sarver et al., 1990, Science247:1222-1225). In another embodiment, the oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

In an alternative embodiment, the Nogo antisense nucleic acid of theinvention is produced intracellularly by transcription from an exogenoussequence. For example, a vector can be introduced in vivo such that itis taken up by a cell, within which cell the vector or a portion thereofis transcribed, producing an antisense nucleic acid (RNA) of theinvention. Such a vector would contain a sequence encoding the Nogoantisense nucleic acid. Such a vector can remain episomal or becomechromosomally integrated, as long as it can be transcribed to producethe desired antisense RNA. Such vectors can be constructed byrecombinant DNA technology methods standard in the art. Vectors can beplasmid, viral, or others known in the art, used for replication andexpression in mammalian cells.

Expression of the sequence encoding the Nogo antisense RNA can be by anypromoter known in the art to act in mammalian, preferably human, cells.Such promoters can be inducible or constitutive. Such promoters includebut are not limited to: the SV40 early promoter region (Bernoist andChambon, 1981, Nature 290:304-310), the promoter contained in the 3′long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981,Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences ofthe metallothionein gene (Brinster et al., 1982, Nature 296:39-42), etc.

The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a Nogo gene,preferably a human Nogo gene. However, absolute complementarity,although preferred, is not required. A sequence “complementary to atleast a portion of an RNA,” as referred to herein, means a sequencehaving sufficient complementarity to be able to hybridize with the RNA,forming a stable duplex; in the case of double-stranded Nogo antisensenucleic acids, a single strand of the duplex DNA may thus be tested, ortriplex formation may be assayed. The ability to hybridize will dependon both the degree of complementarity and the length of the antisensenucleic acid. Generally, the longer the hybridizing nucleic acid, themore base mismatches with a Nogo RNA it may contain and still form astable duplex (or triplex, as the case may be). One skilled in the artcan ascertain a tolerable degree of mismatch by use of standardprocedures to determine the melting point of the hybridized complex.

5.8.2.1.2 Therapeutic Use of Nogo Antisense Nucleic Acids

The Nogo antisense nucleic acids can be used to treat (or prevent)disorders of a cell type that expresses, or preferably overexpresses,Nogo. In a specific embodiment, such a disorder is a growthproliferative disorder. In a preferred embodiment, a single-stranded DNAantisense Nogo oligonucleotide is used.

Cell types which express or overexpress Nogo RNA can be identified byvarious methods known in the art. Such methods include but are notlimited to hybridization with a Nogo-specific nucleic acid (e.g. byNorthern hybridization, dot blot hybridization, in situ hybridization),observing the ability of RNA from the cell type to be translated invitro into Nogo, immunoassay, etc. In a preferred aspect, primary tissuefrom a patient can be assayed for Nogo expression prior to treatment,e.g., by immunocytochemistry or in situ hybridization.

Pharmaceutical compositions of the invention, comprising an effectiveamount of a Nogo antisense nucleic acid in a pharmaceutically acceptablecarrier, can be administered to a patient having a disease or disorderwhich is of a type that expresses or overexpresses Nogo RNA or protein.

The amount of Nogo antisense nucleic acid which will be effective in thetreatment of a particular disorder or condition will depend on thenature of the disorder or condition, and can be determined by standardclinical techniques. Where possible, it is desirable to determine theantisense cytotoxicity of the tumor type to be treated in vitro, andthen in useful animal model systems prior to testing and use in humans.

In a specific embodiment, pharmaceutical compositions comprising Nogoantisense nucleic acids are administered via liposomes, microparticles,or microcapsules. In various embodiments of the invention, it may beuseful to use such compositions to achieve sustained release of the Nogoantisense nucleic acids. In a specific embodiment, it may be desirableto utilize liposomes targeted via antibodies to specific identifiabletumor antigens (Leonetti et al., 1990, Proc. Natl. Acad. Sci. U.S.A.87:2448-2451; Renneisen et al., 1990, J. Biol. Chem. 265:16337-16342).

5.9 Demonstration of Therapeutic or Prophylactic Utility

The Therapeutics of the invention are preferably tested in vitro, andthen in vivo for the desired therapeutic or prophylactic activity, priorto use in humans. For example, in vitro assays which can be used todetermine whether administration of a specific Therapeutic is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered aTherapeutic, and the effect of such Therapeutic upon the tissue sampleis observed. For example, a Therapeutic that is an inhibitor of Nogofunction can be assayed by measuring neurite regrowth or functionalrecovery of motor control in the patient.

In various specific embodiments, in vitro assays can be carried out withrepresentative cells of cell types involved in a patient's disorder, todetermine if a Therapeutic has a desired effect upon such cell types.

Compounds for use in therapy can be tested in suitable animal modelsystems prior to testing in humans, including but not limited to rats,mice, chicken, cows, monkeys, rabbits, etc. For in vivo testing, priorto administration to humans, any animal model system known in the artmay be used.

5.10 Therapeutic/Prophylactic Administration and Compositions

The invention provides methods of treatment (and prophylaxis) byadministration to a subject of an effective amount of a Therapeutic ofthe invention. In a preferred aspect, the Therapeutic is substantiallypurified. The subject is preferably an animal, including but not limitedto animals such as cows, pigs, horses, chickens, cats, dogs, etc., andis preferably a mammal, and most preferably human. In a specificembodiment, a non-human mammal is the subject.

Formulations and methods of administration that can be employed when theTherapeutic comprises a nucleic acid are described above; additionalappropriate formulations and routes of administration can be selectedfrom among those described hereinbelow.

Various delivery systems are known and can be used to administer aTherapeutic of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe Therapeutic, receptor-mediated endocytosis (see, e.g., Wu and Wu,1987, J. Biol. Chem. 262:4429-4432), construction of a Therapeuticnucleic acid as part of a retroviral or other vector, etc. Methods ofintroduction include but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds may be administered by any convenient route,for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local. Inaddition, it may be desirable to introduce the pharmaceuticalcompositions of the invention into the central nervous system by anysuitable route, including intraventricular and intrathecal injection;intraventricular injection may be facilitated by an intraventricularcatheter, for example, attached to a reservoir, such as an Ommayareservoir. Pulmonary administration can also be employed, e.g., by useof an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion during surgery, topical application, e.g., inconjunction with a wound dressing after surgery, by injection, by meansof a catheter, or by means of an implant, said implant being of aporous, non-porous, or gelatinous material, including membranes, such assialastic membranes, or fibers. In one embodiment, administration can beby direct injection at the site (or former site) of a malignant tumor orneoplastic or pre-neoplastic tissue.

In another embodiment, the Therapeutic can be delivered in a vesicle, inparticular a liposome (see Langer, Science 249:1527-1533 (1990); Treatet al., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp. 353-365 (1989);Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)

In yet another embodiment, the Therapeutic can be delivered in acontrolled release system. In one embodiment, a pump may be used (seeLanger, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, N.Y.(1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61(1983); see also Levy et al., Science 228:190 (1985); During et al.,Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)).In yet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., the brain, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

In a specific embodiment where the Therapeutic is a nucleic acidencoding a protein Therapeutic, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptidc which is knownto enter the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad.Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid Therapeuticcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of aTherapeutic, and a pharmaceutically acceptable carrier. In a specificembodiment, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. Oral formulation can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,etc. Examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositionswill contain a therapeutically effective amount of the Therapeutic,preferably in purified form, together with a suitable amount of carrierso as to provide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The Therapeutics of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed with freeamino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

The amount of the Therapeutic of the invention which will be effectivein the treatment of a particular disorder or condition will depend onthe nature of the disorder or condition, and can be determined bystandard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. However, suitable dosage ranges forintravenous administration are generally about 20-500 micrograms ofactive compound per kilogram body weight. Suitable dosage ranges forintranasal administration are generally about 0.01 pg/kg body weight to1 mg/kg body weight. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

5.11 Diagnosis and Screening

Nogo proteins, analogues, derivatives, and subsequences thereof, Nogonucleic acids (and sequences complementary thereto), anti-Nogoantibodies, have uses in diagnostics. Such molecules can be used inassays, such as immunoassays, to detect, prognose, diagnose, or monitorvarious conditions, diseases, and disorders affecting Nogo expression,or monitor the treatment thereof. In particular, such an immunoassay iscarried out by a method comprising contacting a sample derived from apatient with an anti-Nogo antibody under conditions such thatimmunospecific binding can occur, and detecting or measuring the amountof any immunospecific binding by the antibody. In a specific aspect,such binding of antibody, in tissue sections, can be used to detectaberrant Nogo localization or aberrant (e.g., low or absent) levels ofNogo. In a specific embodiment, antibody to Nogo can be used to assay ina patient tissue or serum sample for the presence of Nogo where anaberrant level of Nogo is an indication of a diseased condition. By“aberrant levels,” is meant increased or decreased levels relative tothat present, or a standard level representing that present, in ananalogous sample from a portion of the body or from a subject not havingthe disorder.

The immunoassays which can be used include but are not limited tocompetitive and non-competitive assay systems using techniques such asimmunohistochemistry, pathology, western blots, radioimmunoassays, ELISA(enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, immunohistochemistry assays, protein A immunoassays, toname but a few.

Nogo genes and related nucleic acid sequences and subsequences,including complementary sequences, can also be used in hybridizationassays. Nogo nucleic acid sequences, or subsequences thereof comprisingabout at least 8 nucleotides, can be used as hybridization probes.Hybridization assays can be used to detect, prognose, diagnose, ormonitor conditions, disorders, or disease states associated withaberrant changes in Nogo expression and/or activity as described supra.In particular, such a hybridization assay is carried out by a methodcomprising contacting a sample containing nucleic acid with a nucleicacid probe capable of hybridizing to Nogo DNA or RNA, under conditionssuch that hybridization can occur, and detecting or measuring anyresulting hybridization.

In specific embodiments, diseases and disorders involving cellulargrowth and development disorders can be diagnosed, or their suspectedpresence can be screened for, or a predisposition to develop suchdisorders can be detected, by detecting decreased levels of Nogoprotein, Nogo RNA, or Nogo functional activity as demonstrated growthinhibition, or by detecting mutations in Nogo RNA, DNA or protein (e.g.,translocations in Nogo nucleic acids, truncations in the Nogo gene orprotein, changes in nucleotide or amino acid sequence relative towild-type Nogo) that cause decreased expression or activity of Nogo.Such diseases and disorders include but are not limited to thosedescribed in Section 3 and Section 5.8.1.1. By way of example, levels ofNogo protein can be detected by immunoassay, levels of Nogo RNA can bedetected by hybridization assays (e.g., Northern blots, dot blots), Nogobinding to cellular growth inhibitor protein receptors can be done bybinding assays commonly known in the art, translocations and pointmutations in Nogo nucleic acids can be detected by Southern blotting,RFLP analysis, PCR using primers that preferably generate a fragmentspanning at least most of the Nogo gene, sequencing of the Nogo genomicDNA or cDNA obtained from the patient, etc.

Kits for diagnostic use are also provided, that comprise in one or morecontainers an anti-Nogo antibody, and, optionally, a labeled bindingpartner to the antibody. Alternatively, the anti-Nogo antibody can belabeled (with a detectable marker, e.g., a chemiluminescent, enzymatic,fluorescent, or radioactive moiety). A kit is also provided thatcomprises in one or more containers a nucleic acid probe capable ofhybridizing to Nogo RNA. In a specific embodiment, a kit can comprise inone or more containers a pair of primers (e.g., each in the size rangeof 6-30 nucleotides) that are capable of priming amplification [e.g., bypolymerase chain reaction (see e.g., Innis et al., 1990, PCR Protocols,Academic Press, Inc., San Diego, Calif.), ligase chain reaction (see EP320,308) use of Qβ replicase, cyclic probe reaction, or other methodsknown in the art] under appropriate reaction conditions of at least aportion of a Nogo nucleic acid. A kit can optionally further comprise ina container a predetermined amount of a purified Nogo protein or nucleicacid, e.g., for use as a standard or control.

5.12 Screening for Nogo Agonists and Antagonists

Nogo nucleic acids, proteins, and derivatives also have uses inscreening assays to detect molecules that specifically bind to Nogonucleic acids, proteins, or derivatives and thus have potential use asagonists or antagonists of Nogo, in particular, molecules that thusaffect cellular growth regulation. In a preferred embodiment, suchassays are performed to screen for molecules with potential utility asneural growth promoters for drug development. The invention thusprovides assays to detect molecules that specifically bind to Nogonucleic acids, proteins, or derivatives. For example, recombinant cellsexpressing Nogo nucleic acids can be used to recombinantly produce Nogoproteins in these assays, to screen for molecules that bind to a Nogoprotein. Molecules (e.g., putative binding partners of Nogo) arecontacted with the Nogo protein (or fragment thereof) under conditionsconducive to binding, and then molecules that specifically bind to theNogo protein are identified. Similar methods can be used to screen formolecules that bind to Nogo derivatives or nucleic acids. Methods thatcan be used to carry out the foregoing are commonly known in the art.

By way of example, diversity libraries, such as random or combinatorialpeptide or nonpeptide libraries can be screened for molecules thatspecifically bind to Nogo. Many libraries are known in the art that canbe used, e.g., chemically synthesized libraries, recombinant (e.g.,phage display libraries), and in vitro translation-based libraries.

Examples of chemically synthesized libraries are described in Fodor etal., 1991, Science 251:767-773; Houghten et al., 1991, Nature 354:84-86;Lam et al., 1991, Nature 354:82-84; Medynski, 1994, Bio/Technology12:709-710; Gallop et al., 1994, J. Medicinal Chemistry 37(9):1233-1251;Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb etal., 1994, Proc. Natl. Acad. Sci. USA 91:11422-11426; Houghten et al.,1992, Biotechniques 13:412; Jayawickreme et al., 1994, Proc. Natl. Acad.Sci. USA 91:1614-1618; Salmon et al., 1993, Proc. Natl. Acad. Sci. USA90:11708-11712; PCT Publication No. WO 93/20242; and Brenner and Lerner,1992, Proc. Natl. Acad. Sci. USA 89:5381-5383.

Examples of phage display libraries are described in Scott and Smith,1990, Science 249:386-390; Devlin et al., 1990, Science, 249:404-406;Christian, R. B., et al., 1992, J. Mol. Biol. 227:711-718); Lenstra,1992, J. Immunol. Meth. 152:149-157; Kay et al., 1993, Gene 128:59-65;and PCT Publication No. WO 94/18318 dated Aug. 18, 1994.

in vitro translation-based libraries include but are not limited tothose described in PCT Publication No. WO 91/05058 dated Apr. 18, 1991;and Mattheakis et al., 1994, Proc. Natl. Acad. Sci. USA 91:9022-9026.

By way of examples of nonpeptide libraries, a benzodiazepine library(see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91:4708-4712)can be adapted for use. Peptoid libraries (Simon et al., 1992, Proc.Natl. Acad. Sci. USA 89:9367-9371) can also be used. Another example ofa library that can be used, in which the amide functionalities inpeptides have been permethylated to generate a chemically transformedcombinatorial library, is described by Ostresh et al. (1994, Proc. Natl.Acad. Sci. USA 91:11138-11142).

Screening the libraries can be accomplished by any of a variety ofcommonly known methods. See, e.g., the following references, whichdisclose screening of peptide libraries: Parmley and Smith, 1989, Adv.Exp. Med. Biol. 251:215-218; Scott and Smith, 1990, Science 249:386-390;Fowlkes et al., 1992; BioTechniques 13:422-427; Oldenburg et al., 1992,Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al., 1994, Cell76:933-945; Staudt et al., 1988, Science 241:577-580; Bock et al., 1992,Nature 355:564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA89:6988-6992; Ellington et al., 1992, Nature 355:850-852; U.S. Pat. Nos.5,096,815, 5,223,409, and 5,198,346, all to Ladner et al.; Rebar andPabo, 1993, Science 263:671-673; and PCT Publication No. WO 94/18318.

In a specific embodiment, screening can be carried out by contacting thelibrary members with a Nogo protein (or nucleic acid or derivative)immobilized on a solid phase and harvesting those library members thatbind to the protein (or nucleic acid or derivative).

Examples of such screening methods, termed “panning” techniques aredescribed by way of example in Parmley and Smith, 1988, Gene 73:305-318;Fowlkes et al., 1992, BioTechniques 13:422-427; PCT Publication No. WO94/18318; and in references cited hereinabove.

In another embodiment, the two-hybrid system for selecting interactingproteins in yeast (Fields and Song, 1989, Nature 340:245-246; Chien etal., 1991, Proc. Natl. Acad. Sci. USA 88:9578-9582) can be used toidentify molecules that specifically bind to a Nogo protein orderivative.

5.13 Animal Models

The invention also provides animal models, including but not limited tomodels in mice, hamsters, sheep, pigs, cattle, and preferably non-humanmammals.

In one embodiment, animal models for diseases and disorders involvingneurite extension, growth and regeneration are provided. Such an animalcan be initially produced by promoting homologous recombination betweena Nogo gene in its chromosome and an exogenous Nogo gene that has beenrendered biologically inactive (preferably by insertion of aheterologous sequence, e.g., an antibiotic resistance gene). In apreferred aspect, this homologous recombination is carried out bytransforming embryo-derived stem (ES) cells with a vector containing theinsertionally inactivated Nogo gene, such that homologous recombinationoccurs, followed by injecting the ES cells into a blastocyst, andimplanting the blastocyst into a foster mother, followed by the birth ofthe chimeric animal (“knockout animal”) in which a Nogo gene has beeninactivated (see Capecchi, 1989, Science 244:1288-1292). The chimericanimal can be bred to produce additional knockout animals. Such animalscan be mice, hamsters, sheep, pigs, cattle, etc., and are preferablynon-human mammals. In a specific embodiment, a knockout mouse isproduced.

Such knockout animals are expected to develop or be predisposed todeveloping diseases or disorders involving the central nervous systemand thus can have use as animal models of such diseases and disorders,e.g., to screen for or test molecules (e.g., potential nervous systemdisorder therapeutics) for the ability to inhibit tumors of nerve tissueand thus treat or prevent such diseases or disorders.

The present invention is not to be limited in scope by the microorganismdeposited or the specific embodiments described herein. Indeed, variousmodifications of the invention in addition to those described hereinwill become apparent to those skilled in the art from the foregoingdescription and accompanying Figures. Such modifications are intended tofall within the scope of the appended claims.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

6. EXAMPLE Characterization of the Nucleotide and Protein Product of theNogo Gene

The examples described herein demonstrate that the cloned gene, Nogo,encodes a protein that is a potent neural cell growth inhibitor and isalso recognized by the monoclonal antibodies described in Schwab et al.,U.S. Pat. No. 5,684,133.

6.1 Materials and Methods

The following sections describe materials and methods used in thepresent invention. One of ordinary skill in the art will recognize thatthese materials and methods are merely illustrative of the presentlyclaimed invention and modifications are envisioned by the presentinventors. Such modifications are intended to fall within the scope ofthe appended claims.

6.1.1 Purification of Bovine Nogo from Myelin

All purification steps were carried out at 4° C. and inhibitorysubstrate activity of the obtained fractions was routinely determined bythe NIH 3T3 spreading and PC12 neurite outgrowth assays (Section6.1.10). Bovine spinal cord tissue was carefully cleaned by strippingoff the meninges and cut into small pieces. The myelin was thenextracted in extraction buffer (60 mM CHAPS, 100 mM Tris-Cl, pH 8.0, 10mM EDTA buffer, pH 8.0, 2.5 mM iodacetamide, 1 mM phenylmethylsulfonylfluoride, 0.1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml peptstatin A).

To obtain spinal cord extract, the tissue was homogenized directly inCHAPS extraction buffer in a ratio of (1:1; w:v). The homogenate wascentrifuged twice at 100,000×g (Kontron type: K50.13, fixed angle) for 1hour at 4° C. The clear supernatant (extract) was immediately applied toa Q-Sepharose column (2.6×11.5 cm), equlibrated in buffer A (20 mMTris-Cl, pH 8.0, 0.5% (w/v) CHAPS). Bound proteins were eluted with afive-bed volume linear gradient from 0 to 1 M NaCl in buffer A (100 mlgradient in 50 minutes). Active fractions containing bovine NI220 elutedaround 0.4 M NaCl and were pooled (q-pool 1) for subsequent applicationson Superdex 200 (2.6×60 cm) column, equilibrated in buffer B (150 mMNaCl, 20 mM Tris-Cl, pH 8.0, 0.5% (w/v) CHAPS).

Active fractions, after gel filtration (s-pool 1), were separated by 6%SDS-PAGE under reducing conditions and low constant power (2 watts/gel)for a total of 2500 Volt-hours. Bands and gel regions were identifiedafter Coomoassie Blue staining (0.1% w/v R250 in 50% methanol and 10%acetic acid), cut out, and extracted in 800 μl of gel elution buffer(0.5% (w/v) CHAPS, 20 mM Tris-Cl, pH 8.0, 10 mM EDTA, pH 8.0, 2.5 mMiodacetamide, 1 mM phenylmethylsulfonyl fluoride, 0.1 μg/ml aprotinin, 1μg/ml leupeptin, 1 μg/ml peptstatin A) for at least 48 hours at 4° C.

6.1.2 Microsequencing of Purified Nogo

The IN-1 neutralizable active gel-eluted material of several gels wasre-run on a 10% SDS-polyacrylamide gel under reducing conditions, andstained with 0.1% (w/v) Coomassie Blue R250 in 50% methanol and 10%acetic acid. The 220 KDa band was cut out, and endoproteinase Lys-Cdigestion (1:50 molar ratio) was performed directly in the gel. Thesample was acidified and applied to a reverse phase high performanceliquid chromatography column, peptides were separated with a lineargradient (0-100%) of 0.04% trifluoroacetic acid and 80% acetonitrile,and fractions containing single peptide species were subjected toautomated Edman degradation.

6.1.3 Electrophoresis of Purified Nogo

High resolution SDS-PAGE was carried out using 6% (w/v)SDS-polyacrylamide gels (10×24×0.01 cm) under reducing conditions (100mM dithiothreitol). Transfer onto Immobilon-P membranes (Millipore) wasperformed in 20 mM Tris base, 192 mM glycine, pH 8.3, 0.037% (w/v) SDS,20% methanol with a semi-dry transfer apparatus (Bio-Rad, Trans BlotSD). Transfer time was 2 h at 0.8 mA/cm². Blocking reagent (1 hour atroom temperature) was 3% gelatin in PBS (phosphate-buffered saline, pH7.2, 8 g NaCl, 0.2 g of kH₂PO₄, 2.8 g of NaHPO₄.12H₂O, and 0.2 g of KCl,dissolved in 1 liter of water) and the washing solution contained 20 mMTris-Cl, pH 7.5, 150 mM NaCl, and 0.4% Tween (3×10 minutes at roomtemperature). Incubation time for the first antibody (for dilution with1% gelatin in PBS) was usually overnight at 4° C. Horseradishperoxidase-conjugated anti-mouse IgG secondary antibody (1:2000) wasincubated for 1 hour at room temperature. The ECL chemiluminescencesystem was used for detection (Amersham Pharmacia Biotech).

6.1.4 cDNA Library Probing

White matter was freshly dissected from bovine spinal cord, and poly(A)⁺RNA was extracted using the FastTrack kit (Invitrogen). Construction ofcDNA libraries was performed using the Uni-ZAP kit (Stratagene)following the manufacture's. instructions. The complexity of thelibraries was greater than 4×10⁶ plaque forming units in total, and theaverage size of the inserts was approximately 1.8 kilobases.

Degenerate oligonucleotides MSC5-8 (MSC5:

-   TCIGTIGGYAAIACIGCIGGYAARTC (SEQ ID NO:47); MSC6:-   TCIGTIGGIAGIACIGCIGGYAAYTC (SEQ ID NO:48); MSC7:-   TCIGTIGGYAAIACIGCIGGIAGRTC (SEQ ID NO:49); MSC8:-   TCIGTIGGIAGIACIGCIGGIAGRTC (SEQ ID NO:50)) were designed from the    bNI220 peptide 1 sequence, and MSC9 (GARATHGCIGAIATHCARGAYGGIGA (SEQ    ID NO:51)) was designed from bNI220 peptide 2 sequence.    Oligonucleotides were synthesized by MWG Biotech (Munchenstein,    Switzerland) and labeled with the DIG DNA 3′-end labeling kit.    Riboprobes were synthesized using the DIG RNA labeling kit    (Boehringer Mannheim).

Probe hybridization and washing conditions were as described by themanufacturer (MSC5-8 and MSC9 were used at a hybridization and washingtemperature of 57° C.). Probe detection was performed using the CDP-starsystem (Boehringer Mannheim). CDNA library handling and screening wasdone according to the protocols for lambda ZAP CDNA libraries(Stratagene). Genescreen (DuPont) nylon membranes were used for plaquelifts.

6.1.5 DNA Sequencing

Both strands of CWP1-3, Oli18, Oli3, and R1-3U21 were sequenced with thePerkin Elmer AB1377 system by Microsynth (Balgach, Switzerland). DNAsequences were analyzed by the DNASIS program (Hitachi). Databasesearches were performed with the BLAST program (NCBI).

6.1.6 RNA Analysis

Total RNA and poly(A)⁺ RNA were extracted from tissues using the RNAgent(Promega) or FastTrack kit (Invitrogen), respectively. RNAs wereseparated by electrophoresis on 1% formaldehyde gels and transferred toGenescreen membranes. Blots were hybridized with antisense riboprobes,which were generated with the DIG RNA labeling kit (BoehringerMannheim), from the relevant plasmids. Blot hybridization, washing, andCDP star-detection conditions were as described by the manufacturer. The‘common’ probe, EST111410 (TIGR, ATCC, Rockville, Md., USA) containstranscript A sequence between nucleotides 2535-4678, the exon 1 specificprobe contains transcript A sequence between nucleotides 65-769, and theexon 2 specific-probe contains transcript A sequence between nucleotides815-3183.

6.1.7 Antisera Production

Antiserum 472 (AS 472) was generated by Research Genetics, Inc.(Huntsville Ala.; USA) against the synthetic peptide P472,SYDSIKLEPENPPPYEEA (bovine sequence; SEQ ID NO:33), which corresponds torat Nogo amino acid sequence at 623 to 640 of SEQ ID NO:2, with threemismatches.

Antiserum Bruna (AS Bruna) was generated against a fragment ofrecombinant Nogo protein, expressed in E. coli as a fusion protein.Specifically, the carboxy-terminus of the rat Nogo A nucleotide sequenceencoding amino acids 762 to 1,163 of SEQ ID NO:2 (expressed in E. coliusing the Novagen pET system) was used to generate AS Bruna anti-Nogoantisera.

6.1.8 Electrophoresis and Western Blotting

SDS-PAGE and Western blotting was performed by standard methods wellknown to those skilled in the art. Antibodies were diluted as follows:AS Bruna 1: 7,500; AS 472 1:2,000; anti-myc (9E10) 1:5,000 (Invitrogen);anti-BiP 2 μg/ml (Stressgen); mAb IN-1 hybridoma supernatant was usedundiluted. Secondary antibodies were: HRP conjugated anti-rabbit(Pierce; 1:20,000); anti-mouse I gM (1:50,000); and alkaline phosphataseconjugated anti-mouse (Milan Analytica AG, La Roche, Switzerland;1:5,000).

6.1.9 Immunohistochemistry

Adult rat spinal cord or cerebellum was rapidly dissected, embedded inOTC compound and frozen at −40° C. Twenty post mortem sections were cutand fixed in ethanol/acetic acid at 40° C. Immunostaining was performedas described by Rubin et al., 1994, J. Neurocytol. 23:209-217, exceptthat the quenching step was omitted. Alternatively, tissue sections werefixed by methanol (2 minutes at −20° C.), and immunostaining was carriedout as per Rubin et al., supra. Primary antibodies used were (Antibody:(dilution)): Hybridoma supernatant of IN-1: (undiluted); AS Bruna:(1:5000); or affinity purified AS 472: (1:50).

6.1.10 NIH 3T3 Fibroblast Spreading Assay

NIH 3T3 fibroblasts were plated onto culture dishes pre-coated with 5μg/well (=1 cm²) q-pool. Q-pool is the pooled active fractions of bovinespinal cord extract separated on a Q sepharose column. IN-1 was used asundiluted culture supernatant (1-10 μg/ml), AS Bruna and pre-immuneserum were diluted 1:1000 in PBS, and AS 472 and pre-immune serum werediluted 1:500 in PBS. To compensate for activity variations in differentq-pool preparations the number of inhibited, round cells plated on theq-pool was normalized to 100% and to 0% plated on buffer control(Spillman et al., 1998, J. Biol. Chem. 273:19283-93).

6.1.11 DRG Neurite Outgrowth Assay

Dorsal root ganglia (DRG)s were dissected from E16 embryonic chicken inHank's balanced salt solution (HBSS), divided into two parts and platedon dishes pre-coated with q-pool in 100 μl F12 medium with 10% (FCS) and1% methocel. Neurite outgrowth from individual DRGs was scored after 24hours incubation at 37° C. in a semi-quantitative way using a scale of 0(no outgrowth) to 4 (maximum outgrowth).

6.1.12 DRG/Optic Nerve Co-culture Assay

Optic nerves were dissected from adult rats, irradiated with 5500 Graysand injected either with AS 472 or the corresponding pre-immune serum(1:10 dilution). Pairs of nerves were cultured in 3-chamber culturessuch that one end of each nerve reached through a silicon grease/teflonring barrier into the middle chamber, where dissociated cultured primaryDRGs neurons from P0 rats were placed. After two weeks in culture, thenerves were fixed by standard techniques known in the art and embeddedfor electron microscopy (EM), and ultrathin sections were taken at adistance of about 3.5 mm from the DRG-exposed stump. Sections weresystematically analyzed for the presence of regrowing axons using aZeiss EM 902.

6.1.13 Nogo a Expression in COS Cells

The Nogo A open reading frame was subcloned into the pcDNA3.1mychisvector (Invitrogen) using standard cloning techniques known in the art.The resulting plasmid (Nogo-myc19) gave rise to a recombinant proteincontaining the Nogo A sequence fused to the myc-his tag (21 aminoacids). Nogo-myc19 (2 μg DNA per 35 mm dish) or control plasmid(pcDNAmychisLacZ) were transfected into COS cells using superfect(Qiagen) according to the manufacturer's protocol. Transfected cellswere harvested 36-48 hours after transfection. Based onimmunofluorescent staining with anti-myc antibody and enzymaticβ-galactosidase color reaction, the average transfection rate wasestimated to be about 20%. Transfected COS cells were fixed with 95%ethanol/5% acetic acid (4° C., 25 minutes), blocked in PBS/10% FCS, andincubated with AS Bruna (1:200) or IN-1 (1:2) for 2 hours in PBS/1% FCSat room temperature. Cells were washed with PBS and reacted withfluorescent secondary antibodies (goat anti-rabbit-FITC for AS Bruna andgoat anti-mouse-TRITC for IN-1 detection, Jackson Immuno Research Lab.Inc., West Grove, Pa.).

6.1.14 Oligodendrocyte Cultures

Oligodendrocytes isolated from new-born rat brain were plated on 75 cm²poly-lysine flasks (Sigma, St. Louis, Mo.) and cultured for 10-12 daysin DMEM supplemented with 5% FCS. Enriched, mixed populations ofoligodendrocytes and their progenitors were released from the astrocytemonolayer by shaking overnight at 210 rpm in an orbital shaker. Thecells were plated at a density of 1-2×10⁶ cells on poly-lysine coated 35cm² dishes. Progenitors were allowed to differentiate in chemicallydefined medium (CDM) for 3-4 days.

6.1.15 Cell Surface Biotinylation

P4 rat whole brain cultures were prepared as described in van der Haar,et al. (1998, J. Neurosci. Res. 51:371-81). At day 7 in vitro they werebiotinylated with the cell-impermeable EZ-LINK-Sulfo-NHS-LC-Biotin(Pierce) as described except that all steps were carried out at 15° C.and cells lysed in 1 ml of lysis buffer (0.05M NaH₂PO₄ pH8.0, 0.15MNaCl, 0.5% CHAPS (Sigma), 2.5 mM iodacetamide, 1 mM phenylmethylsulfonylfluoride, 0.1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A).Biotinylated proteins were immuno-precipitated with Dynabeads M-280Streptavidin (Dynal) subjected to SDS-PAGE and transferred tonitrocellulose membranes that were probed with AS472, α-BiP andα-β-tubulin. The membranes were stripped with Re-Blot Western BlotRecycling Kit (Chemicon).

6.1.16 Immunocytochemistry

Optic nerve oligodendrocytes were prepared as described in Schwab andCaroni (1988, J. Neurosci. 8:2381-2393). Two day-old cultures wereincubated with AS 472 (1:200) or mAb IN-1 (1:3) in medium for 25 minutesat room temperature (rt). Cultures were washed, fixed with 4%paraformaldehyde/5% sucrose in PBS, and blocked in 0.1M maleic acid/2%blocking reagent (Boehringer Mannheim) for 1 hour. Secondary alkalinephosphatatase conjugated antibodies (Milan Analytica) were used at1:7,500 in 0.1M maleic acid/1% blocking reagent (1 hour, rt).Transfected COS cells were fixed with 95% ethanol/5% acetic acid (4° C.,25 minutes), blocked, and incubated with AS Bruna (1:200) or mAb IN-1for 2 hours at rt. Cells were reacted with goat anti-rabbit-FITC, andgoat anti-mouse TRITC (Jackson Immuno Research Lab).

6.1.17 Optic Nerve Chamber

Pairs of optic nerve were cultured in a 3-chamber culture system asdescribed in Schwab et al. (1988, J. Neurosci. 8:2381-2393), injectedwith and exposed to either AS 472 or the corresponding pre-immune serum(1:10). Optic nerves were embedded for electron microscopy (EM), andultra-thin sections were taken at a distance of about 3.5 mm from theDRG-exposed stump. Sections were systematically analysed for thepresence of regrowing axons using a Zeiss EM 902 microscope.

6.2 Experimental Results

The following section discloses the experimental results obtained fromthe methods sections set forth in 6.1 and subsections.

6.2.1 Isolation of Nogo CDNA

The bovine homologue of rat NI-250 was purified, bNI220, and peptides ofthe purified protein were generated by protease digestion. Multipledigoxygenin-labeled degenerate oligonucleotides were designed accordingto six different bNI220 peptide sequences. Several cDNA clones wereisolated from the screening of a bovine white matter library using theseoligonucleotides. The insert of the longest clone (CWP1-3, FIG. 1 a) wasused to synthesize probes for subsequent screening of rat cDNAlibraries. Selected clones from such screenings are shown in FIG. 1 a.DNA sequence analysis of these cDNA clones suggested that threedifferent transcripts originate from one gene, and this gene wasdesignated Nogo. The different transcripts likely result from bothalternative promoter usage and alternative splicing (Nogo A, Nogo B andNogo C; FIG. 1 b). DNA sequences were compiled from the clones shown inFIG. 1A to create the transcript A, the DNA sequence of which is shownin FIG. 2 a.

Conceptual translation of the three transcripts gives rise to proteinproducts designated Nogo A (1163 amino acids), Nogo B (360 amino acids)and Nogo C (199 amino acids). Since Nogo A contains all six peptidesequences obtained from purified bNI220 (FIG. 2 b), it is likelyequivalent to the purified protein, rat NI-250. Nogo A, B, and C have acommon carboxy terminus of 188 amino acids (the common domain), and NogoA and B share an amino terminus of 172 amino acids. Nogo A is longerthan Nogo B by 803 amino acids due to alternative splicing.

None of the Nogo isoforms possess a hydrophobic stretch of amino acidsat the N-terminus, which could be used as a conventional signal peptide.However, proteins have been described which lack a conventional signalpeptide but are still transferred through membranes, for examplefibroblast growth factor (Florkiewicz et al., 1995, J. Cell. Physiology162:388-399), ciliary neurotrophic factor (Sendtner et al., 1994, J.Neurobiology 25:1436-1353) and interleukin-1 (Rubartelli et al., 1990,EMBO J. 9:1503-1510). Membrane proteins such as commissureless (Tear etal., 1996, Neuron 16:501-514) also lack a conventional signal peptideyet are inserted into the membrane.

Although there is no in-frame stop codon in the putative 5′-untranslatedregion which would unequivocally define the start codon, the followingevidence suggests that the methionine indicated in FIG. 2 a is the startcodon for Nogo A and Nogo B: (1) The sequence around this presumed startcodon conforms well with the consensus sequence for translation startsites (GCCGCC A/G CCATGG; SEQ ID NO:39); (2) Extensive efforts were madeto search for more upstream sequences by both library screening and5′-RACE. None of these searches have resulted in the identification ofmore upstream sequences; and (3) Eukaryotic recombinant Nogo A expressedfrom the above mentioned methionine has an apparent molecular weight ofabout 200 kD, as estimated by SDS-PAGE, which is indistinguishable fromendogenous Nogo A from rat oligodendrocytes (FIG. 11 a).

6.2.2 Nogo Sequence Analysis

Nogo A contains seven potential N-glycosylation sites, howeverbiochemical evidence indicates Nogo A does not have a majorpolysaccharide component. Nogo A also has nineteen recognition sites forPKC, and seven recognition sites for casein kinase II (FIG. 2 a). Allthree Nogos have two common carboxy terminal hydrophobic domains of 35and 36 amino acids, respectively. Either or both of them may be used astrans- or intra-membrane domains, which is consistent with thecharacterization of bNI220 as an integral membrane protein. Nogo A (aswell as Nogo B and C) does not contain any motifs of known cell adhesionmolecules, extracellular matrix proteins, or other guidance molecules.

Nogo sequences were used to search different databases for homologousgenes, the carboxy terminals common domain of the three Nogo products issimilar (62.5%) to an identified human gene, nsp (cl13 and s-rex in rat,and chs-rex in chicken) (FIG. 3). An EST from C. elegans and aDrosophila melanogaster EST also have significant similarity (16.6% and13.6%, respectively) to both Nogo and nsp at this same region. The 180amino acid carboxy terminal domains of both Nogos and Nsps are highlyconserved across mammalian species (98.3% and 97.3%, respectively),which suggests that they may perform similar and essential functions.Outside of this region, the similarity for a given protein among speciesis also high (73% between rat and bovine Nogo A; 76.2% between NSP-A andS-rexb; 50% between Chs-rexb and NSP-A or S-rexb). The similaritybetween NSPs and Nogos are, however, limited to their carboxy terminal,hydrophobic, common domain (FIG. 3 a), and to the acidic nature of theproteins outside of this conserved region. The NSPs (NSP-A, -B and -C)have been previously described as neuro-endocrine specific products withunknown functions. In situ hybridization and immunohistology showed aneuronal localization of NSPs in the nervous system. Another human gene,nsp-like-1, which also has a carboxy terminal hydrophobic region with50% similarity to both Nsp and Nogo was recently identified.

6.2.3 Nogo Tissue Expression

The Nogo expression pattern was examined by Northern blotting and insitu hybridization. When a “common” probe (Section 6.1.6) was used,three major Nogo transcripts (Designated: A, 4.6 kb; B, 2.6 kb; and C,1.7 kb) were detected in the optic nerve, the spinal cord, and thecerebral cortex (FIG. 4 a). In dorsal root ganglia, only the two largertranscripts were detected. A 2.6 kb, major transcript was detected inPC12 cell, whereas a 4.6 kb band can be detected only after longexposures (FIG. 4 a). In sciatic nerve, lower levels of transcripts weredetected with the 2.6 kb band being the major transcript. When spinalcord and PC 12 cell poly (A)⁺ RNA were hybridized to an exon 1 specificprobe, only the 4.6 kb and the 2.6 kb transcripts were detected; whenthe hindbrain and skeletal muscle poly(A)⁺ RNAs were hybridized to anexon 2-specific probe, only the 4.6 kb transcript in hindbrain wasdetected (FIG. 4 b). These results verify the transcript map shown inFIG. 1B. Northern blotting results, however, also demonstrated that Nogoexpression is not restricted to the nervous system (FIG. 4 c); Nogotranscripts were also detected in skeletal muscle (1.7 kb), kidney (2.6kb and 1.7 kb), cartilage (from the breastbone, 1.7 kb), skin (1.7 kb),lung (2.6 kb), and spleen (2.6 kb). Except for the skeletal muscle,which expresses Nogo C transcripts at a high level, the level of Nogotranscripts outside of the nervous system is lower than that of thenervous system. So far, the 4.6 kb Nogo A transcripts seem to beuniquely transcribed in the nervous system.

In situ hybridizations on adult rat CNS tissue sections using the commonprobe showed moderate labeling of rows of cell bodies in the whitematter of various parts of the brain and the spinal cord. Thisarrangement is typical of interfascicular oligodendrocytes (FIG. 5 a,d). In addition to oligodendrocytes, several types of neurons alsoexpress Nogo transcripts at high levels (FIG. 5 c, e). In cerebellum,double staining of sections with an anti-GFAP antibody and in situhybridization clearly showed a strong labeling of the Purkinje cells bythe Nogo probe, whereas astrocytes were not labeled (FIG. 5 e, f). Indeveloping optic nerves, Nogo transcripts were detected as early aspostnatal day 0 (P0), i.e., several days before the mRNAs of the majormyelin proteins proteolipid protein (PLP) and myelin basic protein (MBP)can be detected (FIG. 6). This timing is consistent with the appearanceof the first galactocerebroside-positive oligodendrocytes and theexpression of a neurite growth inhibitory activity, which can beneutralized by IN-1.

Antisera were generated against a synthetic peptide based on a bovineNogo A specific sequence (AS 472), and against a 45 kD recombinant,partial rat Nogo A (AS Bruna) (Section 6.1.7). AS 472 and AS Bruna each,recognized a protein of about 200 kD in bovine myelin, and AS Brunafurther recognized a 200 kD rat myelin protein on Western blots (FIG.7). Sections of adult rat spinal cord and cerebellum were stained withAS 472, AS Bruna, and IN-1. When the sections were fixed withethanol/acetic acid (a procedure shown earlier to be required forpreservation and accessibility of IN-1 antigens) strong staining ofwhite matter/myelin was seen with all three antibodies (FIG. 8).Staining of oligodendrocyte cell bodies was particularly distinct withAS Bruna. Treatment of the fresh frozen sections with methanol insteadof ethanol/acetic acid abolished the myelin stain except foroligodendrocyte cell bodies.

AS Bruna and AS 472 also stained some types of neurons including motorneurons in spinal cord, and granular and molecular layers in thecerebellum. Purkinje cells stained strongly with AS 472 and AS Bruna,but there was no detectable staining with IN-1.

6.2.4 Nogo Antibodies Inhibit Nogo Induced Growth Inhibition in Vitro

Semi-purified bovine spinal cord NI-220 preparations (q-pool) canprevent NIH 3T3 fibroblast spreading and neurite outgrowth. In thepresence of Nogo antisera, either AS Bruna, AS 472, or IN-1, the q-poolinhibitory activity was reduced, i.e., NIH 3T3 fibroblasts underwentspreading and embryonic chicken dorsal root ganglia (DRG) extendneurites on dishes coated with q-pool (FIG. 9). Specificity wasdemonstrated by addition of peptide P472, which was the peptide used toraise AS 472 (Section 6.1.7). P472 successfully blocked the inhibitoryeffect of AS 472, whereas, a control peptide had no effect on theinhibition.

Furthermore, the presence of Nogo A on the cell surface ofoligodendrocytes was demonstrated immunocytochemically, functionally andbiochemically using AS 472. When live, primary cultured oligodendrocyteswere stained with either mAb IN-1 or AS 472 a relatively weak (ascompared to immunocytochemistry for galactocerebroside) but clearsurface staining was observed on differentiated oligodendrocytes (FIG. 5a, c). Addition of the competing peptide (P472) for AS 472 or omittingthe primary antibody abolished the specific staining (FIG. 15 b, d).Cell surface biotinylation and subsequent precipitation withstreptavidin further proved the presence of Nogo A on the plasmamembrane of oligodendrocytes. In the precipitate, AS 472 detected a bandrunning about 40 kD above the intracellular, probably non-processed andnon-glycosylated AS 472 immuno-positive band. The ER protein BiP couldnot be detected in the biotinylated fraction (FIG. 15 e).

Nogo A as an oligodendrocyte cell surface molecule was also analyzedfunctionally. Co-culturing of oligodendrocytes and NIH 3T3 fibroblastsor oligodendrocytes and DRG neurons showed clearly the inhibitoryproperties of mature oligodendrocytes. These assays demonstrate that NIH3T3 fibroblasts and DRG neurites strongly avoided the territory of theoligodendrocytes, an effect that was neutralized by mAb IN-1. In thepresence of AS 472, this inhibition was equally reduced (FIGS. 16 a, band e, f), while preincubation of AS 472 with P472 restored theoligodendrocyte-mediated inhibition (FIGS. 16 c, d and g, h).Quantification revealed the highly significant neutralizing capacity ofmAb IN-1 and AS 472 in both types of assays (FIGS. 16 i and j).

RecNogo-A (FIG. 17 a) produced by a stably transfected CHO cell line wastested for its activity on NIH 3T3 fibroblast spreading and DRG neuriteoutgrowth. Recombinantly produced β-galactosidase isolated from a stableCHO cell line (CHO-LacZ) was enriched in parallel with recNogo-A and wasused as a control for inhibitory activity of endogenous CHO proteins inboth assays. In the NIH 3T3 fibroblast spreading assay,recNogo-A-containing CHO extract (Nogo-A: about 1-5% of total protein;FIG. 9 a) exhibited clear inhibitory effects on cell spreading at 10μg/cm² (FIG. 17 b). This effect was dose-dependent: at 20 μg/cm² theinhibitory activity was higher, whereas 5 μg/cm² was not inhibitory(data not shown). The inhibitory activity could be neutralized tobackground levels by preincubation of the coated protein with mAb IN-1or AS Bruna, whereas a control antibody against galactocerebroside (mAbO1) or AS Bruna preimmune serum had no effect (FIG. 17 b).

In addition to its strong effect on the NIH 3T3 fibroblast spreading,recNogo-A-containing CHO extract, but not CHO-LacZ extract had a potentinhibitory effect on neurite outgrowth from primary cultured neurons:Dissociated DRG neurons were inhibited by recNogo-A in a dose-dependentmanner (FIG. 17 c). This inhibitory activity could be neutralized by mAbIN-1, but not by the control mAb O1 (FIG. 17 c-e). Recombinant proteinisolated from CHO-LacZ was not inhibitory at 1 and 5 μg, and addition ofmAbs O1 or IN-1 had no effect on neurite outgrowth.

6.2.5 Neurite Regrowth In Vitro

The ability of new-born rat DRG neurites to regenerate and grow throughadult CNS tissue was investigated. Pairs of optic nerves were dissectedfrom adult rats and cultured in a special chamber culture system suchthat DRG neurites had access to one end of each nerve (FIG. 10 a). Ineach culture, one of the two nerves was injected with and exposed topre-immune rabbit serum, the second nerve was injected with AS 472,which was also present in the side chamber around the nerve. Followingtwo weeks in vitro in the presence of NGF, the cultures were fixed,disassembled, and embedded for electron microscopy (EM). EM sectionswere taken about 3.5 mm from the end of the nerve in contact with DRGneurons. Pre-immune serum injected nerves contained no or only a fewaxons (FIG. 10 b). The latter were exclusively found associated withbasement membranes and astrocytes at the surface of the nerves. Incontrast, the majority of the optic nerves injected with AS 472contained considerable numbers of axons, often up to several hundred.Contact with myelin could frequently be seen (FIGS. 10 c, d).

6.2.6 Recombinant Nogo a Recognition by IN-1

When Nogo A was expressed as a carboxy terminal myc-his-taggedrecombinant protein in transfected-COS cells, western blotting usingboth anti-myc antibody and AS Bruna demonstrated that the recombinantNogo A has an apparent molecular weight of about 200 kD on denaturingSDS gels (FIG. 11 a). On the same blot, a band of similar mobility wasdetected by AS Bruna in rat primary culture oligodendrocytes, suggestingthat recombinant Nogo A has a nearly identical molecular weight as theendogenous Nogo A from oligodendrocytes (FIG. 1 a). When transfected COScells were stained by immunofluorescence with IN-1 and AS Bruna, IN-1and AS Bruna recognized the same, transfected cells (FIGS. 11 b, c). Themajority of the immunoreactivity was localized intracellularly and wasaccessible only after permeabilization.

6.2.7 Mapping the Nogo Active Region(s)

A series of deletion mutants of Nogo was generated in order to map theinhibitory domain(s) or region(s) of Nogo. The deletion constructs ofthe Nogo gene were generated by using internal restriction sites,exonuclease III-mung bean digestions and polymerase chain reactions. Adescription of the mutants is provided in FIG. 18 and its BriefDescription. The majority of constructs have an N-terminal T7-tag foridentification with anti-T7 monoclonal antibodies; and an N- orC-terminal hexahistidine tag (“His-tag”) for purification usingimmobilised-Co(II)-affinity chromatography. The Nogo deletion mutants,named NiG-D1, NiG-D2, up to NiG-D20, were all tested using the NIH 3T3fibroblast-spreading assay to determine inhibitory activity. Somemutants were tested in PC12 neurite outgrowth, dissociated rat DRGneurite outgrowth or retinal ganglion stripe assays. The results areshown in Table 2 below.

TABLE 2 Functional Activities of Nogo Deletion Mutants 3T3 PC12 DRG RGCNogo-A ++ + + Nogo-B + Nogo-C − − NiAext ++ + + EST +/− NiR + NiG ++ +NiG-D1 + NiG-D2 + NiG-D3 ++ NiG-D4 + NiG-D5 − NiG-D7 + NiG-D8 + NiG-D9 +NiG-D10 +/− NiG-D14 − NiG-D15 + NiG-D16 + NiG-D17 + NiG-D18 + NiG-D20 ++

A positive result in the NIH 3T3 fibroblast assay (3T3) or PC12 assay isscored when fibroblasts or PC12 cells are inhibited from spreading on aplate coated with a preparation of Nogo obtained from a deletion mutant.A positive result in the embryonic chicken dorsal root ganglion neuriteoutgrowth assay (DRG) or ganglion growth cone collapse assay (RGC)indicates that neurite outgrowth is inhibited or that the growth cone iscaused to collapse in the presence of a preparation of Nogo obtainedfrom a deletion mutant.

The data indicate that a major inhibitory domain was identified in theNogo-A specific region from amino acid numbers 172-974, particularlyamino acid numbers 542-722. In addition, the N-terminal sequence ofNogo-A and Nogo-B (amino acid numbers 1-171) was also inhibitory for 3T3spreading. Based on the results, regions of Nogo from amino acid numbers172-259, and from numbers 975-1162 appear to be non-essential and can beremoved without loss of inhibitory activity.

7. EXAMPLE Human Nogo Nucleic Acids and Proteins, Derivatives andFragments

The instant invention provides the nucleotide sequences encoding humanNogo protein, and fragments of human Nogo proteins, including the humanequivalents to rat Nogo A, Nogo B and part of Nogo C. The human Nogoamino acid sequence is depicted in FIG. 13 and has been assigned SEQ IDNO:29.

The instant invention also provides nucleotide sequences of fragments ofthe human Nogo gene. The human Nogo nucleotide sequence can bedetermined using the rat Nogo A transcript as an aid to align and splicetogether human expressed sequence tags (EST) that are homologous to therat or bovine cDNA sequence.

For example, the ESTs AA081783 and AA333267 (Section 5.1) overlap witheach other and correspond to rat Nogo A (FIG. 2 a; SEQ ID NO:1) nucleicacid positions 765 to 1272. The ESTs AA322592, AA092565, and AA081525(Section 5.1) also overlap with each other and the overlapping sequencescorrespond to rat Nogo nucleic acids 1642 to 2131. These two independentsets of overlapping ESTs cannot be aligned to give the human sequencewithout direct computer comparison to the rat or bovine Nogo nucleotidesequence of the present invention. For the initial computer alignment,ENTREZ Nucleotide QUERY is preferable. Other computer alignment programsare listed in Section 5.1, as alternative examples and are not meant tolimit the scope of computer programs that can be used.

8. Discussion 8.1 Cloning of the Neurite Growth Inhibitor Nogo

Nogo A has many properties that support it being the previouslydescribed rat NI-250, a major neurite outgrowth inhibitory protein ofCNS myelin and the antigen of the IN-1. At the molecular level, Nogo Acontains all six peptides originally obtained by sequencing of bNI-220,the most inhibitory component of bovine spinal cord myelin. At theexpression level, oligodendrocytes are the major cell type in adult CNSfor Nogo A expression, and the timing of Nogo expression in the opticnerve matches the previous description of a IN-1 neutralized myelininhibitory activity for neurite growth. Moreover, Western blottingrevealed the presence of Nogo A in active q-pool fractions, and whitematter from various CNS regions was stained with AS Bruna as well as AS472 (specific for Nogo A) in a pattern identical to that of IN-1. Bothof these facts agree with the interpretation that Nogo A is NI-250.

Two antisera raised against Nogo A sequences, AS Bruna and AS 472,greatly decreased the inhibitory activity of a partially purified bovinespinal cord preparation (q-pool). AS 472 also allowed ingrowth of largenumbers of dorsal root ganglia axons over several millimeters into adultoptic nerve explants, very similar to IN-1.

Although the calculated molecular weight of Nogo A is about 140 kD, ithas an apparent molecular weight of about 200 kD on denaturing SDS gels,which is in the range of the previous estimation of about 250 kD. Theaberrant mobility of Nogo A in SDS gels is likely caused by its acidicnature rather than post-translational modifications. Aberrant mobilityof proteins on SDS-PAGE has been postulated for other highly acidicproteins such as the Growth-Associated Protein GAP-43, as well as NSP-A.Furthermore, bacterially expressed recombinant Nogo A has the sameapparent molecular weight as that of the endogenous Nogo A expressed byrat oligodendrocytes. This argues against major modifications of Nogo Aby mechanism such as glycosylation.

8.2 Nogo Prevents Regeneration and Restricts Plasticity of the Adult CNS

The expression of Nogo in rat optic nerve oligodendrocytes from P0 onagrees well with the earlier findings of a IN-1 neutralizable neuritegrowth inhibitory activity. Interestingly, this expression precedes thatof the main myelin proteins, and compact myelin formation by severaldays. The appearance of Nogo, possibly in response to axonal signals,could prevent further axon growth in the corresponding fiber tracts(axon numbers peak at E20 in rat optic nerves). Nogo could also inhibitcollateral formation and thereby stabilize the general structure of thedifferentiated CNS. In grey matter of different CNS regions, the contentof myelin and IN-1 immunoreactivity correlates inversely with the levelof GAP-43 and the plastic potential of the given regions. Indeed, IN-1antibodies applied to the adult CNS allow sprouting and plasticity tooccur in the brainstem and the spinal cord to an extent known previouslyonly of the new-born CNS. The large functional recovery that parallelsthis plasticity indicates that sprouting axons are able to formfunctionally appropriate connections.

It has been demonstrated previously that the response of neurons toinhibitory Nogos differs between neurons of different ages. Presumably,this is due to a differential expression of receptors, which willhopefully soon be characterized. Like for the Netrins and many growthfactors the existence of different Nogo receptors, which triggerdifferent responses, remains a possibility. The fact that Nogos are alsoexpressed in some types of neurons points to possible interactionsbetween the same and/or different Nogo isoforms.

8.3 Nogo Belongs to a New Family of Neurite Regulatory Molecules

Sequence analysis of Nogo reveals no known motifs of cell surface ormatrix proteins involved in axonal guidance (repulsive or attractive);i.e. no Immunoglobulin, fibronectin type III, or EGF-domains could beidentified. Neither was there homology to described neural growthinhibitors, the Semaphorins, the Netrins, or the Ephrins.

Nogos form a novel family with a group of recently described proteins,the NSP/s-rex and the NSP-like 1 proteins, based on the similarity oftheir carboxy terminal 180 amino acids. Like in the case of Nogo, bothalternative promoter usage (both Nsp and Nsp-like 1 gene) andalternative splicing (Nsp only) are responsible for the production ofalternative protein products with common carboxy termini containing twostretches of hydrophobic amino acids. As indicated by the name, the NSPs(neuroendocrine-specific proteins) are predominantly expressed inneurons and several endocrine cell types. They localize mainlyintracellularly in association with the endoplasmic reticulum. TheNSP-like 1 gene is expressed predominantly in brain and muscle. Thefunctions of neither the NSP nor the Nsp-like 1 families are known. Thefact that potential orthologs exist in both C. elegans and Drosophilamelanogaster suggests that Nogo, with its nerve regeneration andsprouting inhibitory activity, may be a newly evolved and heretoforeundescribed member of the NSP family.

8.4 Nogo in Non-neuronal Tissues

The Nogo C transcript is expressed in skeletal muscle at a levelcomparable to that of the nervous system. One conceivable function ofmuscle Nogo C is to repel motor axons and to restrict them to the motorendplate region. Low levels of Nogo expression can also be detected inother non-nervous tissues. The observed inhibition of fibroblast andastrocyte spreading by myelin extract and NI-250 alludes to the presenceof receptors and response mechanisms for Nogo proteins in these cells.This suggests a possible general function of Nogos in contact inhibitionof cell movement.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. A method of immunizing a non-human animal in order to generateantibodies against a Nogo protein, said method comprising administeringto said animal an immunogenic amount of a protein consisting of: (i) thecarboxy terminal 188 amino acids of SEQ ID NO:29; (ii) an amino acidsequence consisting of amino acids 988-1023 of SEQ ID NO:2; (iii) anamino acid sequence consisting of amino acids 975-1162 of SEQ ID NO:2;(iv) an amino acid sequence consisting of amino acids 172 to 974 of SEQID NO:2; (v) an amino acid sequence consisting of amino acids 172 to 723of SEQ ID NO:2; (vi) an amino acid sequence consisting of amino acids542 to 722 of SEQ ID NO:2; (vii) an amino acid sequence consisting ofamino acids 1-171 of SEQ ID NO:2; (viii) an amino acid sequenceconsisting of amino acids 1-974 of SEQ ID NO:2; (ix) an amino acidsequence consisting of amino acids 1-131 of SEQ ID NO:2; (x) an aminoacid sequence consisting of amino acids 680-939 of SEQ ID NO:29; (xi) anamino acid sequence consisting of amino acids 940-1127 of SEQ ID NO:29;(xii) an amino acid sequence consisting of amino acids 259-542 of SEQ IDNO:2; (xiii) an amino acid sequence consisting of amino acids 172-259 ofSEQ ID NO:2; (xiv) an amino acid sequence consisting of amino acids722-974 of SEQ ID NO:2.
 2. A method of immunizing a non-human animal,said method comprising administering to said animal an immunogenicamount of a protein consisting of: (i) a first amino acid sequence inwhich more than 95% of the amino acid residues in said first sequenceare identical to the amino acid residues of a second amino acid sequencein an alignment, said second amino acid sequence consisting of the aminoacid sequence of amino acids 1-131 of SEQ ID NO:29, (ii) a first aminoacid sequence in which more than 95% of the amino acid residues in saidfirst sequence are identical to the amino acid residues of a secondamino acid sequence in an alignment, said second amino acid sequenceconsisting of the amino acid sequence of amino acids 132-939 of SEQ IDNO:29, (iii) a first amino acid sequence in which more than 95% of theamino acid residues in said first sequence are identical to the aminoacid residues of a second amino acid sequence in an alignment, saidsecond amino acid sequence consisting of the amino acid sequence ofamino acids 206-501 of SEQ ID NO:29, (iv) a first amino acid sequence inwhich more than 95% of the amino acid residues in said first sequenceare identical to the amino acid residues of a second amino acid sequencein an alignment, said second amino acid sequence consisting of the aminoacid sequence of amino acids 501-680 of SEQ ID NO:29, (v) a first aminoacid sequence in which more than 95% of the amino acid residues in saidfirst sequence are identical to the amino acid residues of a secondamino acid sequence in an alignment, said second amino acid sequenceconsisting of the amino acid sequence of amino acids 132-206 of SEQ IDNO:29, (vi) a first amino acid sequence in which more than 95% of theamino acid residues in said first sequence are identical to the aminoacid residues of a second amino acid sequence in an alignment, saidsecond amino acid sequence consisting of the amino acid sequence ofamino acids 680-939 of SEQ ID NO:29, or (vii) a first amino acidsequence in which more than 95% of the amino acid residues in said firstsequence are identical to the amino acid residues of a second amino acidsequence in an alignment, said second amino acid sequence consisting ofthe amino acid sequence of amino acids 940-1127 of SEQ ID NO:29, andwherein said protein is free of all central nervous system myelinmaterial, wherein said protein has one or more Nogo functionalactivities selected from the group consisting of: (i) ability to preventregeneration of neurons in the spinal cord or brain; (ii) ability toconfer to a substrate the property of restricting growth, spreading, andmigration of neural cells; (iii) ability to inhibit dorsal root ganglianeurite outgrowth; (iv) ability to block NIH 3T3 cell spreading invitro; and (v) ability to block PC12 neurite outgrowth.
 3. A method ofobtaining polyclonal antibodies to a protein, wherein said proteinconsists of: (i) a first amino acid sequence in which more than 95% ofthe amino acid residues in said first amino acid sequence are identicalto the amino acid residues of a second amino acid sequence in analignment, said second amino acid sequence consisting of the amino acidsequence of amino acids 1-131 of SEQ ID NO:29, (ii) a first amino acidsequence in which more than 95% of the amino acid residues in said firstamino acid sequence are identical to the amino acid residues of a secondamino acid sequence in an alignment, said second amino acid sequenceconsisting of the amino acid sequence of amino acids 132-939 of SEQ IDNO:29, (iii) a first amino acid sequence in which more than 95% of theamino acid residues in said first amino acid sequence are identical tothe amino acid residues of a second amino acid sequence in an alignment,said second amino acid sequence consisting of the amino acid sequence ofamino acids 206-501 of SEQ ID NO:29, (iv) a first amino acid sequence inwhich more than 95% of the amino acid residues in said first amino acidsequence are identical to the amino acid residues of a second amino acidsequence in an alignment, said second amino acid sequence consisting ofthe amino acid sequence of amino acids 501-680 of SEQ ID NO:29, (v) afirst amino acid sequence in which more than 95% of the amino acidresidues in said first amino acid sequence are identical to the aminoacid residues of a second amino acid sequence in an alignment, saidsecond amino acid sequence consisting of the amino acid sequence ofamino acids 132-206 of SEQ ID NO:29, (vi) a first amino acid sequence inwhich more than 95% of the amino acid residues in said first amino acidsequence are identical to the amino acid residues of a second amino acidsequence in an alignment, said second amino acid sequence consisting ofthe amino acid sequence of amino acids 680-939 of SEQ ID NO:29, or (vii)a first amino acid sequence in which more than 95% of the amino acidresidues in said first amino acid sequence are identical to the aminoacid residues of a second amino acid sequence in an alignment, saidsecond amino acid sequence consisting of the amino acid sequence ofamino acids 940-1127 of SEQ ID NO:29, and wherein the protein has one ormore Nogo functional activities selected from the group consisting of:(i) ability to prevent regeneration of neurons in the spinal cord orbrain; (ii) ability to confer to a substrate the property of restrictinggrowth, spreading, and migration of neural cells; (iii) ability toinhibit dorsal root ganglia neurite outgrowth; (iv) ability to block NIH3T3 cell spreading in vitro; and (v) ability to block PC12 neuriteoutgrowth; and wherein said method comprises: (a) immunizing an animalwith said protein that is free of all central nervous system myelinmaterial; and, (b) recovering polyclonal antibodies from the animal. 4.The method of any one of claims 1, 2 and 3, wherein the animal is amouse.
 5. The method of any one of claims 1, 2 and 3, wherein saidprotein is recombinant.
 6. The method of any one of claims 1, 2 and 3,wherein said protein is mammalian.
 7. The method of any one of claims 1,2 and 3, wherein said protein is human.